Maturation of hepatocyte-like cells derived from human pluripotent stem cells

ABSTRACT

The present invention relates to directed differentiation and maturation of hepatocyte-like cells. In particular, the present invention relates to exposure of hepatocyte-like cells to an activator of a retinoic acid responsive receptor, such as retinoic acid (RA), optionally in combination with an inhibitor of GSK-3 (Glycogen synthase kinase 3) or activator of Wnt signalling and/or with the overlay of the cells with one or more components characteristic of the mammalian extracellular matrix (matrix overlay). The present invention also relates to exposure of hepatocyte-like cells to an activator of a retinoic acid responsive receptor, such as retinoic acid (RA), optionally in combination with an inhibitor of a cycline dependent kinase (CDK) and/or with the overlay of the cells with one or more components characteristic of the mammalian extracellular matrix (matrix overlay). The hepatocyte-like cells obtained in accordance with the present invention show a phenotype which is more similar to that of primary hepatocytes than previously shown.

TECHNICAL FIELD

The present invention relates to directed differentiation and maturationof hepatocyte-like cells. The hepatocyte-like cells obtained inaccordance with the present invention show a phenotype which is moresimilar to that of primary hepatocytes than previously shown. Inparticular, the present invention relates to exposure of hepatocyte-likecells to an activator of a retinoic acid responsive receptor, such asretinoic acid (RA), optionally in combination with an inhibitor of GSK-3(Glycogen synthase kinase 3) or activator of Wnt signalling and/or withthe overlay of the cells with one or more components characteristic ofthe mammalian extracellular matrix (matrix overlay). The presentinvention also relates to exposure of hepatocyte-like cells to anactivator of a retinoic acid responsive receptor, such as retinoic acid(RA), optionally in combination with an inhibitor of a cyclin dependentkinase (CDK) and/or with the overlay of the cells with one or morecomponents characteristic of the mammalian extracellular matrix (matrixoverlay). The inventors have, as disclosed herein, found that exposinghepatocyte-like cells to an activator of a retinoic acid responsivereceptor leads to the development of more mature and functional featuresfor the hepatocyte-like cells as well as to more pure and homogenouspopulations of hepatocyte-like cells, compared to currently availablestate of the art methods.

BACKGROUND OF THE INVENTION

The development of novel pharmaceuticals faces a number of challenges,not least the problem of overcoming adverse toxicological effects.Indeed, adverse liver reactions remain the most prominent side effect.Metabolism and ultimate clearance of the majority of small moleculedrugs occurs in the liver, and thus one of the main areas of focus indrug development concerns whether such compounds or their metabolitespossess any hepatotoxic effect. Moreover, it is also of paramountimportance to discover whether the secondary metabolites of suchcompounds also display any cytotoxic effects before the drug can beginclinical trial programmes.

Accordingly there is an urgent need for a model hepatic system thatmimics human liver cells and that is able to predict effects ofcandidate molecules in the development of new drugs or chemicals.Traditionally, researchers have been forced to rely on primaryliver-derived hepatocytes for such screening but these have a number ofserious drawbacks including difficulty of maintaining the cells in longterm culture and difficulty of obtaining consistent, homogeneous cellpopulations. A solution to this has been offered in the form ofhepatocyte-like cells derived from human pluripotent stem cells. Humanpluripotent stem cells (hPS) have already begun to revolutionise theways in which relevant human cell types can be obtained. The possibilityto indefinitely propagate pluripotent human embryonic-derived stem (hES)cells and human induced pluripotent stem (hiPS) cells and subsequentlydifferentiate them into the desired target cell types is now providing astable and virtually unlimited supply of cells for a range ofapplications in vivo and in vitro.

Unfortunately, currently available hepatocyte cell types do not alwaysaccurately model the hepatic environment, due to differences inmorphology and function. For example, one often used alternative toprimary cells are hepatic cell lines which often contain very low levelsof (or totally lack) metabolising enzymes and have expression of otherimportant proteins substantially different from the native hepatocyte invivo. This is of particular relevance in relation to drug metabolismsince one of the major deficiencies in hepatic cell lines is the absenceor abnormally high expression of drug transporter proteins which areessential for drug screening purposes. Other available hepatic celllines suffer from having a morphology and physiology which is morereminiscent of fetal or juvenile hepatocytes than the more clinicallyrelevant adult hepatocytes. For these reasons there is a strong need todevelop hepatocyte cell lines which are not only easy to culture andpropagate but which also possess a more mature phenotype and whichbehave in a manner more akin to adult primary hepatocytes.

Derivation of hepatocyte-like cells from pluripotent stem cells is wellestablished in the art. For in vitro purposes, several groups havedeveloped protocols for deriving hepatocyte-like cells from hES cells(Hay et al., 2007; Hay et al., 2008; Brolen et al. 2010; Funakoshi etal. 2011) as well from hiPS cells (U.S. Pat. No. 8,148,151B; Song et al.2009; Sullivan et al. 2010; Si-Tayeb et al. 2010; Chen et al. 2012).However, common to all of these is a specific low mRNA and proteinexpression of genes typical for mature hepatocytes, like phase I and IIgenes (e.g. CYP1A2, 2B6, 2C9, 2D6, 3A4), nuclear receptors (e.g. CAR andPXR), and other adult hepatic markers (e.g. Albumin). In addition, thesehESC- and hiPSC-derived hepatocyte-like cells have high expression offetal hepatic genes like α-fetoprotein (AFP) and CYP3A7, with the resultthat the cell types described therein have a fetal and not adultphenotype (for overview see e.g. Baxter et al. 2010). Furthermore, inmost of the published studies on hESC- and hiPSC-derived hepatocyte-likecells, expression and functionality of drug transporters has not beeninvestigated at all.

The modulation of RA signalling has been previously shown to be ofimportance during early hepatocyte differentiation and in particular atthe stage when definitive endoderm (DE) is specified to become hepaticendoderm (Touboul et al 2010). Furthermore, RA-response elements havebeen identified in a number of genes important during early hepatocytespecification such as AFP and HNF4α (see Qian et al 2000; Magee et al1998 and Hatzis et al 2001). However, at this early stage RA is known tohave diverse effects and has also been found to be important in thederivation of pancreatic endoderm from pluripotent stem cells (Mfopou etal 2010). US Patent Application Publication US2012/0143316A1 disclosesthe use of all-trans retinoic acid in inducing hepatic differentiationfrom endoderm-like cells. As becomes evident, all of these disclosuresrelate to the modulation of RA signalling during endodermal and earlyhepatocyte differentiation. However, none of these documents teaches orsuggests the applicability of retinoic acid as an hepatocyte maturationpromoting agent, let alone its use at a late stage in hepatocytedifferentiation.

The use of GSK 3 inhibitors have previously been described for earlydifferentiation towards endoderm. WO08094597 (Dalton) describes a methodof producing mesendoderm from primate pluripotent stem cells (pPSC) bycontacting the pPSC with an effective amount of GSK3 inhibitor in adifferentiation media. WO2007050043 (Stanton) describes a method forproducing a mesodermal or an endodermal cell from a pluripotent stemcell, comprising a Wnt-signalling pathway in the pluripotent stem cell.US2006003446 (Keller) describes a way of making a cell populationenriched for endoderm cells culturing embryonic stem cells in theabsence of serum and in the presence of activin and an inhibitor ofWnt-signalling. Modulation of Wnt signalling through the use of a GSK3inhibitor has also been shown to be beneficial in specifying hepatocytecell fate when DE cells are exposed to this treatment (WO2011/116930).Again, all of these disclosures relate to modulation of GSK3 signallingat relatively early stages in endodermal or hepatic specification.

Culturing of cells on certain matrix components has been known to affecttheir growth and, in the case of multipotent cells, to affect theirultimate differentiation. For example, pluripotent stem cells have beenshown to undergo epithelial to mesenchymal transition and thence developinto cardiac cell types through overlaying of the stem cells withcertain matrix components (WO2011060342). Moreover, culturing of adultprimary hepatocytes in defined “sandwich” of matrix components has longbeen known to help them maintain their phenotype and metabolic activity(Dunn et al 1991), (Page et al 2007).

SUMMARY OF THE INVENTION

Present invention describes improved methods by which hepatocyte-likecells derived from human pluripotent stem (hPS) cells, such as but notlimited to hiPS-cells and hES-cells, may be further matured intohepatocyte-like cells possessing a phenotype more closely resemblingthat of ex vivo primary liver hepatocytes.

The present invention provides in a first aspect a method for promotingthe maturation of human hepatocyte-like cells whereby saidhepatocyte-like cells are exposed to an activator of a retinoic acidresponsive receptor, such as retinoic acid, optionally in combinationwith exposure to an inhibitor of GSK3 signalling or activator of Wntsignalling and/or with an overlay of the cells with one or morecomponents characteristic of the mammalian extracellular matrix (matrixoverlay).

Thus, a method for promoting the maturation of human hepatocyte-likecells is provided, the method comprising:

Exposing said human hepatocyte-like cells to an activator of a retinoicacid responsive receptor.

The method for promoting the maturation of human hepatocyte-like cellsmay further comprise culturing human hepatic progenitor cells underdifferentiation conditions to obtain said hepatocyte-like cells.

The present invention provides in a second aspect a method for producinghuman hepatocyte-like cells whereby human hepatic progenitor cells arecultured under differentiation conditions to obtain hepatocyte-likecells, and the obtained hepatocyte-like cells are exposed to anactivator of a retinoic acid responsive receptor, such as retinoic acid,optionally in combination with exposure to an inhibitor of GSK3signalling or activator of Wnt signalling and/or with an overlay of thecells with one or more components characteristic of the mammalianextracellular matrix (matrix overlay).

Thus, a method for producing human hepatocyte-like cells is provided,the method comprising:

Culturing human hepatic progenitor cells under differentiationconditions to obtain hepatocyte-like cells, and

Exposing said hepatocyte-like cells to an activator of a retinoic acidresponsive receptor.

The present inventors have surprisingly found that the exposure ofhepatocyte-like cells to an activator of a retinoic acid responsivereceptor, such as retinoic acid, improves the gene and proteinexpression of a number of markers for mature hepatocytes, notably adultisoforms of HNF4a, CYP1A2, CYP2B6, CYP2C9, CYP3A4, CYP3A5, CAR, GSTA1-1,and NTCP, and thus leads to hepatocyte-like cells with a phenotype moreclosely resembling that of primary hepatocytes. Moreover, a surprisingsynergistic effect was found for exposure to an activator of a retinoicacid responsive receptor, a GSK3-inhibitor and an overlay with one ormore components characteristic of the mammalian extracellular matrixmaking the phenotype of the hepatocyte-like cells even more similar tohuman primary hepatocytes. In addition to improved expression of hepaticgenes and functions, the morphology of the hepatocyte-like cells isimproved, e.g. the cell-cell contacts are enhanced, and the life span ofthe hepatocyte-like cells is prolonged by 7-10 days (FIG. 8).

The activator of a retinoic acid responsive receptor, such as retinoicacid, may be present throughout the differentiation of the hepaticprogenitor cells into hepatocyte-like cells and further maturation ofthe obtained hepatocyte-like cells (“differentiation and maturation”),which may take up to 35 days. Thus, the differentiating and maturinghepatic cells may be continuously/long term exposed to the activator ofa retinoic acid responsive receptor during the differentiation andmaturation. Alternatively, the hepatocyte-like cells may be exposed tosaid activator of a retinoic acid responsive receptor for a continuousperiod of time longer than 4 hours and no longer than 72 hours, such as,e.g., for a continuous period of 5, 24 or 48 hours. The hepatocyte-likecells may also be exposed to said activator of a retinoic acidresponsive receptor for at least two, such as at least three, at leastfour or at least 5, continuous periods of time longer than 4 hours andno longer than 72 hours, such for continuous periods of 5, 24 or 48hours. The at least two continuous periods of time are normallyseparated by a period of non-exposure to said activator of a retinoicacid responsive receptor. Such period of non-exposure may have aduration from several hours to several days, such as from 12 to 24 hoursor 1 to 10 day, such as from 1 to 2 days. In this context, the activatorof a retinoic acid responsive receptor may be added to the culturemedium at any time point during the maturation of the hepatocyte-likecells. The hepatocyte-like cells may be exposed to the activator of aretinoic acid responsive receptor at a time t≥7 days after initiation ofthe differentiation of hepatic progenitor cells into hepatocyte-likecells. Thus, hepatocyte-like cells may be exposed to the activator of aretinoic acid responsive receptor at day 7, 9 or 12 after initiation ofthe differentiation of hepatic progenitor cells into hepatocyte-likecells.

The methods of the present invention may also comprise the initialgeneration of hepatic progenitor cells by culturing hPS cells underdifferentiation conditions (also referred herein as “initial hepaticdifferentiation”). Thus, hPS cells are initially differentiated intosaid hepatic progenitor cells. This initial culturing or differentiationmay include the culturing of the hPS cells under differentiationconditions to obtain cells of the definitive endoderm (DE cells)(pre-endodermal step), and further culturing the obtained DE cells underdifferentiation conditions to obtain hepatic progenitor cells(pre-hepatic step). Accordingly, hPS cells may thus be firstdifferentiated into definitive endoderm, followed by the furtherdifferentiation of the definitive endoderm into hepatic progenitorcells.

Further, during the initial differentiation of hPS cells into endodermaland/or hepatic progenitor cells, the differentiating hPS cells may beexposed to a DNA demethylating agent, such as 5-aza-2-deoxycytidine or5-azacytidine, to demethylate sections of the genome and allowtranscriptional activation of genes. The exposure to said DNAdemethylating agent may take place during the differentiation of the hPScells into DE cells, i.e. during the pre-endodermal step. The cells arethen cultured through endodermal stage until hepatic progenitor stage isreached, i.e. until hepatic progenitor cells are obtained, at whichpoint the further differentiation and maturation of hepatocyte-likecells including the exposure to the activator of a retinoic acidresponsive receptor, either alone or in combination with GSK-3inhibition or activation of Wnt signalling and/or matrix overlay, iscarried out.

The treatment of differentiating hPS cells with a DNA demethylatingagent has surprisingly been found to lead to an improved morphology andyield of DE cells. Moreover, the exposure to the demethylating agentprovides for more pure and homogenous DE populations with lowerexpression of stem cell markers like Oct4, compared to currentlyavailable state of the art methods (see FIG. 13 A to D). Further, anincreased gene expression of a number of markers characteristic fordefinitive endoderm, such as sox17, cxcr4 and hhex (see FIG. 13 D), isseen for these endodermal cells. It is believed to be the first timethat such effects are shown for DNA demethylation and the application ofgrowth factors involved in the differentiation of hPS cells towardsdefinitive endoderm whose action at a genomic level may be enhanced bythe widespread absence of methylation. Moreover, a strong synergisticeffect on the maturation of hepatocyte-like cells is seen when treatingcells with a DNA demethylating agent during early endodermal developmentbefore exposing the obtained hepatic progenitor cells to an activator ofa retinoic acid responsive receptor, either alone or in combination witha GSK3-inhibitor and/or matrix overlay.

As a result of the methods according to the present invention,hepatocyte-like cells are obtained having a phenotype more closelyresembling that of primary hepatocytes. Analysis of cells subsequent tothe maturation period reveals a distinct increase in the expressionlevels of certain markers for mature hepatocytes, notably but notlimited to adult isoforms of HNF4α, CYP1A2, CYP2B6, CYP2C9, CYP3A4,CYP3A5, CAR, GSTA1-1, and NTCP (see for example FIGS. 1B, 4B+C, 6, 9,10, 12). Moreover, in contrast to primary hepatocytes, thehepatocyte-like cells obtained by an early stage demethylation treatmentand late stage exposure to an activator of a retinoic acid responsivereceptor, a GSK3-inhibitor (or activator of Wnt signalling) and a matrixoverlay display a stable or increasing expression of hepatic genes likeCYPs over time in culture. In cultured primary hepatocytes, CYP activityand mRNA expression is rapidly decreasing over time whereas the oppositeis observed for hepatocyte-like cells according to the invention (seeFIG. 16). Another widely used hepatic cell type are HepG2 which displaymuch lower CYP activity than hepatocyte-like cells according to theinvention.

The present invention provides a method for promoting the maturation ofhuman hepatocyte-like cells whereby said hepatocyte-like cells areexposed to an activator of a retinoic acid responsive receptor, such asretinoic acid, optionally in combination with exposure to an CDKinhibitor and/or with an overlay of the cells with one or morecomponents characteristic of the mammalian extracellular matrix (matrixoverlay).

Thus, a method for promoting the maturation of human hepatocyte-likecells is provided, the method comprising:

Exposing said human hepatocyte-like cells to an activator of a retinoicacid responsive receptor.

The method for promoting the maturation of human hepatocyte-like cellsmay further comprise culturing human hepatic progenitor cells underdifferentiation conditions to obtain said hepatocyte-like cells.

The present invention further provides a method for producing humanhepatocyte-like cells whereby human hepatic progenitor cells arecultured under differentiation conditions to obtain hepatocyte-likecells, and the obtained hepatocyte-like cells are exposed to anactivator of a retinoic acid responsive receptor, such as retinoic acid,optionally in combination with exposure to an CDK inhibitor and/or withan overlay of the cells with one or more components characteristic ofthe mammalian extracellular matrix (matrix overlay).

Thus, a method for producing human hepatocyte-like cells is provided,the method comprising:

Culturing human hepatic progenitor cells under differentiationconditions to obtain hepatocyte-like cells, and

Exposing said hepatocyte-like cells to an activator of a retinoic acidresponsive receptor.

As noted above, the present inventors have surprisingly found that theexposure of hepatocyte-like cells to an activator of a retinoic acidresponsive receptor, such as retinoic acid, improves the gene andprotein expression of a number of markers for mature hepatocytes,notably adult isoforms of HNF4α, CYP1A2, CYP2B6, CYP2C9, CYP3A4, CYP3A5,CAR, GSTA1-1, and NTCP, and thus leads to hepatocyte-like cells with aphenotype more closely resembling that of primary hepatocytes. Moreover,a surprising synergistic effect was found for exposure to an activatorof a retinoic acid responsive receptor, a CDK inhibitor and an overlaywith one or more components characteristic of the mammalianextracellular matrix making the phenotype of the hepatocyte-like cellseven more similar to human primary hepatocytes. In addition to improvedexpression of hepatic genes and functions, the morphology of thehepatocyte-like cells is improved, e.g. the cell-cell contacts areenhanced, and the life span of the hepatocyte-like cells is prolonged by7-10 days (FIG. 8).

Again, the activator of a retinoic acid responsive receptor, such asretinoic acid, may be present throughout the differentiation of thehepatic progenitor cells into hepatocyte-like cells and furthermaturation of the obtained hepatocyte-like cells (“differentiation andmaturation”), which may take up to 35 days. Thus, the differentiatingand maturing hepatic cells may be continuously/long term exposed to theactivator of a retinoic acid responsive receptor during thedifferentiation and maturation. Alternatively, the hepatocyte-like cellsmay be exposed to said activator of a retinoic acid responsive receptorfor a continuous period of time longer than 4 hours and no longer than72 hours, such as, e.g., for a continuous period of 5, 24 or 48 hours.The hepatocyte-like cells may also be exposed to said activator of aretinoic acid responsive receptor for at least two, such as at leastthree, at least four or at least 5, continuous periods of time longerthan 4 hours and no longer than 72 hours, such for continuous periods of5, 24 or 48 hours. The at least two continuous periods of time arenormally separated by a period of non-exposure to said activator of aretinoic acid responsive receptor. Such period of non-exposure may havea duration from several hours to several days, such as from 12 to 24hours or 1 to 10 day, such as from 1 to 2 days. In this context, theactivator of a retinoic acid responsive receptor may be added to theculture medium at any time point during the maturation of thehepatocyte-like cells. The hepatocyte-like cells may be exposed to theactivator of a retinoic acid responsive receptor at a time t≥7 daysafter initiation of the differentiation of hepatic progenitor cells intohepatocyte-like cells. Thus, hepatocyte-like cells may be exposed to theactivator of a retinoic acid responsive receptor at day 7, 9 or 12 afterinitiation of the differentiation of hepatic progenitor cells intohepatocyte-like cells.

The methods of the present invention may also comprise the initialgeneration of hepatic progenitor cells by culturing hPS cells underdifferentiation conditions (also referred herein as “initial hepaticdifferentiation”). Thus, hPS cells are initially differentiated intosaid hepatic progenitor cells. This initial culturing or differentiationmay include the culturing of the hPS cells under differentiationconditions to obtain cells of the definitive endoderm (DE cells)(pre-endodermal step), and further culturing the obtained DE cells underdifferentiation conditions to obtain hepatic progenitor cells(pre-hepatic step). Accordingly, hPS cells may thus be firstdifferentiated into definitive endoderm, followed by the furtherdifferentiation of the definitive endoderm into hepatic progenitorcells.

Further, during the initial differentiation of hPS cells into endodermaland/or hepatic progenitor cells, the differentiating hPS cells may beexposed to a DNA demethylating agent, such as 5-aza-2-deoxycytidine or5-azacytidine, to demethylate sections of the genome and allowtranscriptional activation of genes. The exposure to said DNAdemethylating agent may take place during the differentiation of the hPScells into DE cells, i.e. during the pre-endodermal step. The cells arethen cultured through endodermal stage until hepatic progenitor stage isreached, i.e. until hepatic progenitor cells are obtained, at whichpoint the further differentiation and maturation of hepatocyte-likecells including the exposure to the activator of a retinoic acidresponsive receptor, either alone or in combination with CDK inhibitionand/or matrix overlay, is carried out.

The treatment of differentiating hPS cells with a DNA demethylatingagent has surprisingly been found to lead to an improved morphology andyield of DE cells. Moreover, the exposure to the demethylating agentprovides for more pure and homogenous DE populations with lowerexpression of stem cell markers like Oct4, compared to currentlyavailable state of the art methods (see FIG. 13 A to D). Further, anincreased gene expression of a number of markers characteristic fordefinitive endoderm, such as sox17, cxcr4 and hhex (see FIG. 13 D), isseen for these endodermal cells. It is believed to be the first timethat such effects are shown for DNA demethylation and the application ofgrowth factors involved in the differentiation of hPS cells towardsdefinitive endoderm whose action at a genomic level may be enhanced bythe widespread absence of methylation. Moreover, a strong synergisticeffect on the maturation of hepatocyte-like cells is seen when treatingcells with a DNA demethylating agent during early endodermal developmentbefore exposing the obtained hepatic progenitor cells to an activator ofa retinoic acid responsive receptor, either alone or in combination witha CDK inhibitor and/or matrix overlay.

As a result of the methods according to the present invention,hepatocyte-like cells are obtained having a phenotype more closelyresembling that of primary hepatocytes. Analysis of cells subsequent tothe maturation period reveals a distinct increase in the expressionlevels of certain markers for mature hepatocytes, notably but notlimited to adult isoforms of HNF4α, CYP1A2, CYP2B6, CYP2C9, CYP3A4,CYP3A5, CAR, GSTA1-1, and NTCP (see for example FIGS. 1B, 4B+C, 6, 9,10, 12). Moreover, in contrast to primary hepatocytes, thehepatocyte-like cells obtained by an early stage demethylation treatmentand late stage exposure to an activator of a retinoic acid responsivereceptor, a CDK inhibitor and a matrix overlay display a stable orincreasing expression of hepatic genes like CYPs over time in culture.In cultured primary hepatocytes, CYP activity and mRNA expression israpidly decreasing over time whereas the opposite is observed forhepatocyte-like cells according to the invention (see FIG. 16). Anotherwidely used hepatic cell type are HepG2 which display much lower CYPactivity than hepatocyte-like cells according to the invention.

Thus, in further aspects, the invention relates to a hepatocyte-likecell(s) obtained by the methods of the invention and to a cellcomposition(s) comprising, or consisting of, said hepatocyte-likecell(s),

In another aspect, the present invention relates to the further use ofthe hepatocyte-like cell(s) or cell composition(s) of the invention inmedicine, in particular regenerative medicine. In other words, of thehepatocyte-like cell(s) or cell composition(s) of the invention are foruse in medicine, in particular for use in regenerative medicine.Particularly, the hepatocyte-like cell(s) or cell composition(s) of theinvention are for use in the prevention and/or treatment of pathologiesand/or disorders caused by tissue degeneration. The hepatocyte-likecell(s) or cell composition(s) of the invention are also for use in theprevention and/or treatment of liver disorders. The hepatocyte-likecell(s) or cell composition(s) of the invention are also for use in theprevention and/or treatment of metabolic pathologies and/or diseases. Assuch the hepatocyte-like cell(s) or cell composition(s) of the inventionmay be used for the manufacture of a medicament or medicinal product,such as in the form of replacement tissue or cell injection, inparticular for the prevention and/or treatment of pathologies and/ordisorders caused by tissue degeneration. The hepatocyte-like cell(s) orcell composition(s) of the invention may also be used for themanufacture of a medicament or medicinal product/or for the preventionand/or treatment of liver disorders. The hepatocyte-like cell(s) or cellcomposition(s) of the invention may be used for the manufacture of amedicament or medicinal product for the prevention and/or treatment ofmetabolic pathologies and/or diseases. Also included in this aspect ofthe invention are methods for treatment of pathologies and/or disordersmentioned herein, comprising the administration of an effective amountof the hepatocyte-like cell(s) or cell composition(s) of the inventionto a subject in need thereof.

In other aspects, the invention provides the further uses of thehepatocyte-like cell(s) or cell composition(s) of the invention inpharmaceutical and toxicological screening, such as drug discoveryprocesses or toxicity testing; for studying drug transporters or drugmetabolizing enzymes, as in vitro models for studying hepatogenesis; andfor studying human hepatoregenerative disorders.

In a further aspect, the invention relates to the use of an activator ofa retinoic acid responsive receptor for maturing human hepatocyte-likecells. Also included in this aspect is the use of an activator of aretinoic acid responsive receptor in combination with an inhibitor ofGSK3 signalling and/or a matrix overlay for maturing humanhepatocyte-like cells. Further included in this aspect is the use of anactivator of a retinoic acid responsive receptor in combination with anactivator of Wnt signalling and/or a matrix overlay for maturing humanhepatocyte-like cells. Also included in this aspect is the use of anactivator of a retinoic acid responsive receptor in combination with aCDK inhibitor and/or a matrix overlay for maturing human hepatocyte-likecells.

In yet a further aspect, the invention relates to kits useful incarrying out the methods of the invention. Included in this aspect arekits which comprise at least one activator of a retinoic acid responsivereceptor and at least one selected from GSK3 inhibitor, activator of Wntsignalling, CDK inhibitor and extracellular matrix (ECM) component orECM component mixture. It is understood that the details given hereinwith respect to the components employed in the methods of the inventionalso apply to the components comprised by the kits of the invention. Inyet a further aspect, the invention relates to compositions. Suchcompositions are particularly useful for maturing human hepatocyte-likecells in accordance with the invention. Included in this aspect arecompositions which comprise at least one activator of a retinoic acidresponsive receptor and at least one selected from GSK3 inhibitor,activator of Wnt signalling and CDK inhibitor. It is understood that thedetails given herein with respect to the components employed in themethods of the invention also apply to the components comprised by thecompositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods for maturing human hepatocyte-like cellsby exposing the cells to an activator of a retinoic acid receptor,either alone or in combination with exposure to an inhibitor of GSK3signalling or an activator of Wnt signalling and/or with overlaying ofthe cells with one or more components of the mammalian extracellularmatrix (matrix overlay). The methods may further comprise culturing ofhuman hepatic progenitor cells in a supportive culture anddifferentiation medium to obtain said hepatocyte-like cells where thecells are exposed to an activator of a retinoic acid responsivereceptor, either alone or in combination with exposure to an inhibitorof GSK3 signalling or activator of Wnt signalling and/or with overlayingof the cells with one or more components of the mammalian extracellularmatrix (matrix overlay).

The invention also provides methods for maturing human hepatocyte-likecells by exposing the cells to an activator of a retinoic acid receptor,either alone or in combination with exposure to a CDK inhibitor and/orwith overlaying of the cells with one or more components of themammalian extracellular matrix (matrix overlay). The methods may furthercomprise culturing of human hepatic progenitor cells in a supportiveculture and differentiation medium to obtain said hepatocyte-like cellswhere the cells are exposed to an activator of a retinoic acidresponsive receptor, either alone or in combination with exposure to aCDK inhibitor and/or with overlaying of the cells with one or morecomponents of the mammalian extracellular matrix (matrix overlay).

The starting material in the present invention may be any cell having ahepatic cell fate, developed to any stage beyond the endodermal stage,such as but not limited to fetal hepatocytes and hepatic progenitorcells.

As outlined above, the present invention provides a method for promotingthe maturation of human hepatocyte-like cells whereby saidhepatocyte-like cells are exposed to an activator of a retinoic acidresponsive receptor, such as retinoic acid, optionally in combinationwith exposure to an inhibitor of GSK3 signalling or activator of Wntsignalling and/or with an overlay of the cells with one or morecomponents characteristic of the mammalian extracellular matrix (matrixoverlay).

The method for promoting the maturation of human hepatocyte-like cellsmay thus be described as comprising the step:

Exposing said human hepatocyte-like cells to an activator of a retinoicacid responsive receptor.

The method for promoting the maturation of human hepatocyte-like cellsmay further comprise the step of culturing human hepatic progenitorcells under differentiation conditions to obtain said hepatocyte-likecells.

The present invention also provides a method for producing humanhepatocyte-like cells whereby human hepatic progenitor cells arecultured under differentiation conditions to obtain hepatocyte-likecells, and the obtained hepatocyte-like cells are exposed to anactivator of a retinoic acid responsive receptor, such as retinoic acid,optionally in combination with exposure to an inhibitor of GSK3signalling or activator of Wnt signalling and/or with an overlay of thecells with one or more components characteristic of the mammalianextracellular matrix (matrix overlay).

The method for producing human hepatocyte-like cells may thus bedescribed as comprising the following steps:

Culturing human hepatic progenitor cells under differentiationconditions to obtain hepatocyte-like cells, and

Exposing said hepatocyte-like cells to an activator of a retinoic acidresponsive receptor.

The present invention also provides a method for promoting thematuration of human hepatocyte-like cells whereby said hepatocyte-likecells are exposed to an activator of a retinoic acid responsivereceptor, such as retinoic acid, optionally in combination with exposureto a CDK inhibitor and/or with an overlay of the cells with one or morecomponents characteristic of the mammalian extracellular matrix (matrixoverlay).

The method for promoting the maturation of human hepatocyte-like cellsmay thus be described as comprising the step:

Exposing said human hepatocyte-like cells to an activator of a retinoicacid responsive receptor.

The method for promoting the maturation of human hepatocyte-like cellsmay further comprise the step of culturing human hepatic progenitorcells under differentiation conditions to obtain said hepatocyte-likecells.

The present invention also provides a method for producing humanhepatocyte-like cells whereby human hepatic progenitor cells arecultured under differentiation conditions to obtain hepatocyte-likecells, and the obtained hepatocyte-like cells are exposed to anactivator of a retinoic acid responsive receptor, such as retinoic acid,optionally in combination with exposure to CDK inhibitor and/or with anoverlay of the cells with one or more components characteristic of themammalian extracellular matrix (matrix overlay).

The method for producing human hepatocyte-like cells may thus bedescribed as comprising the following steps:

Culturing human hepatic progenitor cells under differentiationconditions to obtain hepatocyte-like cells, and

Exposing said hepatocyte-like cells to an activator of a retinoic acidresponsive receptor.

Human hepatic progenitor cells may thus be used as starting materialaccording to the invention. The hepatic progenitor starting materialmay, for example, be an established cell line of hepatic progenitorcells or may be prepared de novo, such as from hPS cells or endodermalcells.

The differentiation and maturation of hepatocyte-like cells may bedivided into two phases, i.e. a first phase where the hepatic progenitorcells differentiate into hepatocyte-like cells (“hepatic progenitorphase”), and a second phase where the obtained hepatocyte-like cellsfurther mature (maturation phase) During the maturation phase theobtained hepatocyte-like cells exhibit an increased gene and proteinexpression of characteristic markers for hepatocytes.

Suitable conditions for differentiating hepatic progenitor cells intohepatocyte-like cells from hES cells (Hay et al., 2007; Hay et al.,2008; Brolen et al. 2010; Funakoshi et al. 2011) and from hiPS cells(U.S. Pat. No. 8,148,151B; Song et al. 2009; Sullivan et al. 2010;Si-Tayeb et al. 2010; Chen et al. 2012) are known. WO 2009/013254 A1,for example, describes suitable basic protocols to obtainhepatocyte-like cells from hepatic progenitor cells (Embodiments 1 to4).

Generally, hepatic progenitor cells are cultured in a differentiationmedium comprising one or more growth factors, such as HGF, and/or one ormore differentiation inducer, such as dimethylsulfoxide (DMSO),dexamethazone (DexM), omeprazole, Oncostatin M (OSM), rifampicin,desoxyphenobarbital, ethanol or isoniazide. The concentration of the oneor more growth factors, such as HGF, is usually in the range of about 10to about 300 ng/ml, such as about 20 to about 250 ng/ml, about 50 toabout 250 ng/ml, about 100 to about 250 ng/ml, about 150 to about 250ng/ml, about 50 to about 200 ng/ml, about 50 to about 150 ng/ml or about50 to about 100 ng/ml; or may be about 100 ng/ml, about 150 ng/ml, about200 ng/ml, about 250 ng/ml or about 300 ng/ml. The concentration of theone or more differentiation inducer may vary depending on the particularcompound used. The concentration of DMSO, for example, is usually in therange of about 0.1 to about 2% v/v, such as about 0.1 to about 1.5% v/v,about 0.1 to about 1% v/v, about 0.25 to about 1% v/v, about 0.25 toabout 0.75% v/v, about 0.5 to about 1.5% v/v, or about 0.5 to about 1%v/v. The concentration of OSM, for example, is usually in the range ofabout 1 to about 20 ng/ml, such as about 1 to about 15 ng/ml, about 5 toabout 15 ng/ml, or about 7.5 to about 12.5 ng/ml. The concentration ofDexM, for example, is usually in the range of about 0.05 to about 1 μM,such as about 0.05 to about 0.5 μM, about 0.05 to about 0.2 μM, about0.05 to about 0.1 μM or about 0.1 to about 0.5 μM.

The differentiation medium may further comprise serum, such as FBS orFCS, and/or one or more bone morphogenetic proteins (BMPs), such as bonemorphogenetic protein 2 (BMP2) and/or bone morphogenetic protein 4(BMP4). The concentration of serum, if present, is usually in the rangeof about 0.1 to about 5% v/v, such as about 0.1 to about 0.5%, 0.2 to 3%v/v, about 0.5 to about 2.5% v/v, about 0.5 to 1% v/v or about 1 toabout 2.5% v/v. The concentration of the one or more BMPs, if present,is usually in the range of about 50 to about 300 ng/ml, such as about 50to about 250 ng/ml, about 100 to about 250 ng/ml, about 150 to about 250ng/ml, about 50 to about 200 ng/ml, about 100 to about 200 ng/ml orabout 150 to about 200 ng/ml.

The differentiation medium may further comprise other supplements suchas PEST and/or GlutaMAX. The concentration of PEST is usually in therange of about 0.1 to about 0.5% v/v, such as about 0.1 to about 0.25%v/v. The concentration of GlutaMAX is usually in the range of about 0.5to about 1.5% v/v, such as about 0.75 to 1.25% v/v, e.g. about 1% v/v.

The culture medium forming the basis for the differentiation medium maybe any culture medium suitable for culturing human hepatic progenitorcells such as RPMI 1640 or advanced medium, Dulbecco's Modified EagleMedium (DMEM), HCM medium, HBM medium, Waymouth medium or Williams Ebased medium. Thus, the differentiation medium may be RPMI 1640 oradvanced medium comprising or supplemented with the above-mentionedcomponents. Alternatively, the differentiation medium may be DMEMcomprising or supplemented with the above-mentioned components. As afurther alternative, the differentiation medium may thus be HCM or HBMmedium comprising or supplemented with the above-mentioned components.As a further alternative, the differentiation medium may thus beWaymouth medium or Williams E based medium comprising or supplementedwith the above-mentioned components.

The differentiation of human hepatic progenitor cells and furthermaturation of the obtained hepatocyte-like cells (“differentiation andmaturation”) normally takes up to 35 days in total. Thus, in order toobtain hepatocyte-like cells, the human hepatic progenitor cells arecultured in differentiation medium for up to 35 days. For example, thehuman hepatic progenitor cells may be cultured in differentiation mediumfor any time between about 7 to about 35 days. They may thus also becultured for about 10 to about 30 days. They may also be cultured forabout 10 to about 25 days. Alternatively, they may be cultured for about10 to about 20 days or for about 10 to about 15 days. They may also becultured for about 15 to about 35 days. Thus, they may also be culturedfor about 15 to about 30 days. Alternatively, they may be cultured forabout 15 to about 25 days. They may also be cultured for about 15 toabout 20 days. During the culturing the differentiation medium isusually exchanged for fresh medium every second or third day.

Under the above described conditions, hepatocyte-like cells are obtainedfrom hepatic progenitor cells on or after 7 days of culture. Thus, thedifferentiation and maturation of hepatocyte-like cells may be dividedinto a hepatic progenitor phase of 7 days, whereby hepatic progenitorcells differentiate into hepatocyte-like cells, and a maturation phaselasting until the end of the total culture period (e.g., until day 35),whereby the obtained hepatocyte-like cells further mature.

The activator of a retinoic acid responsive receptor employed in themethods of the invention may be any compound capable of binding to andactivating a human retinoic acid receptor (RAR) and/or human retinoid Xreceptor (RXR), such as, e.g., a compound capable of binding to andactivating both RAR and RXR.

A suitable activator of a retinoic acid responsive receptor for use inthe invention is retinoic acid, such as 9-cis-retinoic acid and13-cis-retinoic acid or other retinoic isomers, includingall-trans-retinoic acid, 7-cis retinoic acid and 11-cis-retinoic acid,or an analogue of retinoic acid, such as TTNPB, AM580, retilloic acid orCBS-211A, or a retinoid.

Accordingly, 9-cis-retinoic acid may be used as the activator of aretinoic acid responsive receptor in accordance with the presentinvention. Alternatively, or in addition, 13-cis-retinoic acid may alsobe used as the activator of a retinoic acid responsive receptor inaccordance with the present invention. 13-cis retinoic acid may also beused as the activator of a retinoic acid responsive receptor inaccordance with the present invention.

9-cis-retinoic acid, for example, has been reported to be the onlyretinoic acid stereoisomer that binds to and activates both RXR and RAR(Allenby et al.; Idres et al.). However, another report stated that also11-cis-retinoic acid, 13-cis-retinoic acid and all trans retinoic acidcan bind to RXR but with much lower affinity than 9-cis-retinoic acid(Heyman et al.). Taken together, these reports suggest that9-cis-retinoic acid may be the major RXR activator compared to other RAisomers. Thus, the activator of a retinoic acid responsive receptor foruse in the present invention may be a retinoic acid, an analogue ofretinoic acid or a retinoid capable of binding to and activating bothRAR and RXR, such as, e.g., 9-cis-retinoic acid or an analogue thereof.

Further, all-trans-retinoic acid, 7-cis retinoic acid, 11-cis retinoicacid or 13-cis retinoic acid may be used as activator of a retinoic acidresponsive receptor. Those isomers have been shown to specifically bindRAR, but not to RXR (Allenby et al.; Idres et al.) or with much loweraffinity to RXR than 9-cis-retinoic acid (Heyman et al). Thus, theactivator of a retinoic acid responsive receptor for use in the presentinvention may be a retinoic acid, an analogue of retinoic acid or aretinoid capable of binding to and activating RAR and not or weakly RXR,such as, e.g. all trans retinoic acid or an analogue ofall-trans-retinoic acid, or 7-cis retinoic acid or an analogue of 7-cisretinoic acid, or 11-cis retinoic acid or an analogue of 11-cis retinoicacid, or 13-cis retinoic acid or an analogue of 13-cis retinoic acid.

The activator of a retinoic acid responsive receptor for use in thepresent invention may also be a retinoid capable of binding to andactivating only RXR and not RAR, such as, e.g. Bexarotene (LGD1069),LG100268 or SR11237.

As noted above, an analogue of retinoic acid, such as, e.g., TTNPB,AM580, retilloic acid or CBS-211A, may also be used as the activator ofa retinoic acid responsive receptor. Thus, the retinoic acid analogueTTNPB may be used as the activator of a retinoic acid responsivereceptor. Alternatively, the retinoic acid analogue AM580 may be used asthe activator of a retinoic acid responsive receptor.

Also envisaged is the use of a small molecule, lipid, polypeptide orprotein, which binds to and activates a human retinoic acid receptor(RAR) and/or retinoid X receptor (RXR), as activator of a retinoic acidresponsive receptor. Non-limiting examples of such compounds are Ch 55,AC 261066, AC 55649, CD1530, CD437, CD3254, AM80, BMS 753, BMS 961,Adapalene, Tazarotene, Docosahexaenoic acid, and Fluorobexarotene, whichare all known agonists of RAR or RXR.

Optionally, the hepatocyte-like cells may be exposed to one or morefurther activators of a retinoic acid responsive receptor. Thus, thehepatocyte-like cells may not only be exposed to one activator of aretinoic acid responsive receptor, but may also be exposure to one ormore further activators of a retinoic acid responsive receptor, such asto a combination of two, three or four of those mentioned above. Thehepatocyte-like cells may, for instance, be exposed to both9-cis-retinoic acid and 13-cis-retinoic acid.

In one aspect of the invention, the differentiating and maturing hepaticcells are continuously/long term exposed to the activator of a retinoicacid responsive receptor during the differentiation and maturation ofthe hepatocyte-like cells. Thus, the activator of a retinoic acidresponsive receptor may be present in the differentiation mediumthroughout the differentiation and maturation period.

The differentiating and maturing hepatic cells may, for example, beexposed to the activator of a retinoic acid responsive receptor for upto about 35 days. They may, for example, be exposed to the activator ofa retinoic acid responsive receptor for about 10 days to about 30 days.They may also be exposed to the activator of a retinoic acid responsivereceptor for about 10 days to about 25 days. They may also be exposed tothe activator of a retinoic acid responsive receptor for about 10 daysto about 20 days. They may also be exposed to the activator of aretinoic acid responsive receptor for about 10 days to about 15 days.They may also be exposed to the activator of a retinoic acid responsivereceptor for about 15 days to about 35 days. They may also be exposed tothe activator of a retinoic acid responsive receptor for about 15 daysto about 30 days. They may also be exposed to the activator of aretinoic acid responsive receptor for about 15 days to about 25 days.They may also be exposed to the activator of a retinoic acid responsivereceptor for about 15 days to about 20 days.

In another aspect of the invention, the hepatocyte-like cells areexposed to the activator of a retinoic acid responsive receptor for acontinuous period of time longer than 4 hours and no longer than 72hours. Thus, the activator of a retinoic acid responsive receptor may beadded to, and thus is present, in the differentiation medium for acontinuous period of time longer than 4 hours and no longer than 72hours during the differentiation and maturation period.

The continuous period of time of exposure may be for about 5 to about 10hours. The continuous period of time of exposure may also be for about 5to about 12 hours. The continuous period of time of exposure may also befor about 5 to about 18 hours. The continuous period of time of exposuremay also be for about 5 to about 24 hours. The continuous period of timeof exposure may also be for about 5 to about 48 hours. Thus, thecontinuous period of time of exposure may be for about 5 hours. Thecontinuous period of time of exposure may also be for about 12 to about18 hours. The continuous period of time of exposure may also be forabout 12 to about 24 hours. The continuous period of time of exposuremay also be for about 12 to about 48 hours. The continuous period oftime of exposure may also be for about 12 to about 72 hours. Thus, thecontinuous period of time of exposure may be for about 12 hours. Thecontinuous period of time of exposure may also be for about 18 to about24 hours. The continuous period of time of exposure may also be forabout 18 to about 48 hours. The continuous period of time of exposuremay also be for about 18 to about 72 hours. Thus, the continuous periodof time of exposure may be for about 18 hours. The continuous period oftime of exposure may also be for about 24 to about 48 hours. Thecontinuous period of time of exposure may also be for about 24 to about72 hours. Thus, the continuous period of time of exposure may be forabout 24 hours. The continuous period of time of exposure may also befor about 48 to 72 hours. Thus, the continuous period of time ofexposure may be for about 48 hours or about 72 hours.

The continuous period of time of exposure may, for example, be for about5, about 10, about 12, about 18, about 24, about 48 or about 72 hours.The continuous period of time of exposure may be for about 5. It mayalso be for about 24. Alternatively, the continuous period of time ofexposure may be for about 48.

As shown, for instance, in Example 4 (FIG. 2), exposing hepatocyte-likecells to an activator of a retinoic acid responsive receptor, here9-cis-retinoic acid, for, e.g., 5, 24 and 48 hours, leads to an increasein CYP1A, CYP2C9 and 3A activities when compared to untreated cells.Further, after exposing hepatocyte-like cells to an activator of aretinoic acid responsive receptor for 5 or 24 hours, an increase of mRNAexpression of the adult hepatic gene CYP3A4 and a strong decrease of thefetal hepatic gene CYP3A7 is immediately observed (see Example 5, FIG.3).

The hepatocyte-like cells may not only be exposed to an activator of aretinoic acid responsive receptor once for continuous period of timelonger than 4 hours and no longer than 72 hours, but may also be exposedto said activator of a retinoic acid responsive receptor for at leasttwo, such as at least three, at least four or at least five, continuousperiods of time longer than 4 hours and no longer than 72 hours, suchas, e.g., for continuous periods of 5, 24 or 48 hours. Thus, thehepatocyte-like cells may, for example, be exposed to an activator of aretinoic acid responsive receptor for two continuous periods of timelonger than 4 hours and no longer than 72 hours. The hepatocyte-likecells may also be exposed to an activator of a retinoic acid responsivereceptor for three continuous periods of time longer than 4 hours and nolonger than 72 hours. The hepatocyte-like cells may also be exposed toan activator of a retinoic acid responsive receptor for four continuousperiods of time longer than 4 hours and no longer than 72 hours. Thehepatocyte-like cells may also be exposed to an activator of a retinoicacid responsive receptor for five continuous periods of time longer than4 hours and no longer than 72 hours.

As shown in Example 4, repeated exposure to an activator of a retinoicacid responsive receptor has a stronger increasing effect on, e.g.,CYP1A and CYP2C9 than a single exposure.

After this continuous period of time of exposure, the differentiationmedium is exchanged with one lacking the activator of a retinoic acidresponsive receptor, and the cultivation of the differentiating cells iscontinued. Thus, in a protocol where hepatocyte-like cells are exposedto an activator of a retinoic acid responsive receptor for at least twocontinuous periods of time of exposure as defined above, the at leasttwo continuous periods of time or exposure are separated by a period ofnon-exposure to said activator of a retinoic acid responsive receptor.Such period of non-exposure may have a duration from several hours toseveral days, such as from 12 to 24 hours or from 1 to 10 day. Theperiod of non-exposure may have a duration of from 1 to 2 days. Theperiod of non-exposure may also have a duration from 1 to 5 days. Theperiod of non-exposure may also have a duration from 2 to 5 days. Theperiod of non-exposure may, for instance, have a duration of 1 day. Theperiod of non-exposure may also have a duration of 2 days. The period ofnon-exposure may, for instance, have a duration of 5 days.

In accordance with the invention, the activator of a retinoic acidresponsive receptor may be added to the differentiation medium at anytime point once hepatocyte-like cells have been obtained, such as, e.g.,after 7, 9, 11, 13, 15, 20, 25 and/or 30 days of culturing.

Thus, the activator of a retinoic acid responsive receptor may, forinstance, be added to the differentiation medium for the continuousperiod of time longer than 4 hours and no longer than 72 hours betweenday 7 and day 30 of the differentiation and maturation, such as, e.g.,between day 7 and day 15. The activator of a retinoic acid responsivereceptor may thus be added to the differentiation medium for thecontinuous period of time longer than 4 hours and no longer than 72hours between day 7 and day 9 of the differentiation and maturation.

Accordingly, the activator of a retinoic acid responsive receptor mayalso be added to the differentiation medium for the continuous period oftime longer than 4 hours and no longer than 72 hours at day 7 of thedifferentiation and maturation. The activator of a retinoic acidresponsive receptor may also be added to the differentiation medium forthe continuous period of time longer than 4 hours and no longer than 72hours at day 9 of the differentiation and maturation. The activator of aretinoic acid responsive receptor may also be added to thedifferentiation medium for the continuous period of time longer than 4hours and no longer than 72 hours at day 11 of the differentiation andmaturation. The activator of a retinoic acid responsive receptor mayalso be added to the differentiation medium for the continuous period oftime longer than 4 hours and no longer than 72 hours at day 13 of thedifferentiation and maturation. The activator of a retinoic acidresponsive receptor may also be added to the differentiation medium forthe continuous period of time longer than 4 hours and no longer than 72hours at day 15 of the differentiation and maturation. The activator ofa retinoic acid responsive receptor may also be added to thedifferentiation medium for the continuous period of time longer than 4hours and no longer than 72 hours at day 20 of the differentiation andmaturation. The activator of a retinoic acid responsive receptor mayalso be added to the differentiation medium for the continuous period oftime longer than 4 hours and no longer than 72 hours at day 25 of thedifferentiation and maturation. The activator of a retinoic acidresponsive receptor may also be added to the differentiation medium forthe continuous period of time longer than 4 hours and no longer than 72hours at day 30 of the differentiation and maturation.

The activator of a retinoic acid responsive receptor may also be addedto the differentiation medium for the continuous period of time longerthan 4 hours and no longer than 72 hours at days 7 and 9 of thedifferentiation and maturation. The activator of a retinoic acidresponsive receptor may also be added to the differentiation medium forthe continuous period of time longer than 4 hours and no longer than 72hours at days 7, 9 and 11 of the differentiation and maturation. Theactivator of a retinoic acid responsive receptor may also be added tothe differentiation medium for the continuous period of time longer than4 hours and no longer than 72 hours at days 1, 6, 9, 11 and 16 of thedifferentiation and maturation. The activator of a retinoic acidresponsive receptor may also be added to the differentiation medium forthe continuous period of time longer than 4 hours and no longer than 72hours at days 7, 9, 11 and 16 of the differentiation and maturation. Theactivator of a retinoic acid responsive receptor may also be added tothe differentiation medium for the continuous period of time longer than4 hours and no longer than 72 hours at days 7, 9, 11, 13 and 16 of thedifferentiation and maturation. The activator of a retinoic acidresponsive receptor may also be added to the differentiation medium forthe continuous period of time longer than 4 hours and no longer than 72hours at days 1, 7, 9, 11 and 16 of the differentiation and maturation.The activator of a retinoic acid responsive receptor may also be addedto the differentiation medium for the continuous period of time longerthan 4 hours and no longer than 72 hours at days 1, 3, 7, 9, 11 and 16of the differentiation and maturation.

The hepatocyte-like cells are generally to be exposed to the activatorof a retinoic acid responsive receptor at a concentration in the rangeof about 0.1 to about 5 μM, such as, e.g, in the range of about 0.5 toabout 1.5 μM.

The hepatocyte-like cells may thus be exposed to the activator of aretinoic acid responsive receptor at a concentration in the range ofabout 0.1 to about 2.5 μM. The hepatocyte-like cells may also be exposedto the activator of a retinoic acid responsive receptor at aconcentration in the range of about 0.1 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration in the range of about 0.1 toabout 1 μM. The hepatocyte-like cells may also be exposed to theactivator of a retinoic acid responsive receptor at a concentration inthe range of about 0.1 to about 0.5 μM. The hepatocyte-like cells mayalso be exposed to the activator of a retinoic acid responsive receptorat a concentration in the range of about 0.1 to about 0.3 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration in the range of about 0.2 toabout 5 μM. The hepatocyte-like cells may also be exposed to theactivator of a retinoic acid responsive receptor at a concentration inthe range of about 0.2 to about 2.5 μM. The hepatocyte-like cells mayalso be exposed to the activator of a retinoic acid responsive receptorat a concentration in the range of about 0.2 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration in the range of about 0.5 toabout 5 μM. The hepatocyte-like cells may also be exposed to theactivator of a retinoic acid responsive receptor at a concentration inthe range of about 0.5 to about 3 μM. The hepatocyte-like cells may alsobe exposed to the activator of a retinoic acid responsive receptor at aconcentration in the range of about 0.5 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration in the range of about 0.5 toabout 2 μM. The hepatocyte-like cells may also be exposed to theactivator of a retinoic acid responsive receptor at a concentration inthe range of about 0.5 to about 1.5 μM. The hepatocyte-like cells mayalso be exposed to the activator of a retinoic acid responsive receptorat a concentration in the range of about 0.5 to about 1 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration in the range of about 0.75to about 5 μM. The hepatocyte-like cells may also be exposed to theactivator of a retinoic acid responsive receptor at a concentration inthe range of about 0.75 to about 3 μM. The hepatocyte-like cells mayalso be exposed to the activator of a retinoic acid responsive receptorat a concentration in the range of about 0.75 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration in the range of about 0.75to about 2 μM. The hepatocyte-like cells may also be exposed to theactivator of a retinoic acid responsive receptor at a concentration inthe range of about 0.75 to about 1.5 μM. The hepatocyte-like cells mayalso be exposed to the activator of a retinoic acid responsive receptorat a concentration in the range of about 0.75 to about 1.25 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration in the range of about 1 toabout 2.5 μM. The hepatocyte-like cells may also be exposed to theactivator of a retinoic acid responsive receptor at a concentration inthe range of about 1 to about 2 μM. The hepatocyte-like cells may alsobe exposed to the activator of a retinoic acid responsive receptor at aconcentration in the range of about 1 to about 1.5 μM.

Thus, the hepatocyte-like cells may be exposed to the activator of aretinoic acid responsive receptor at a concentration of about 0.1 μM.The hepatocyte-like cells may also be exposed to the activator of aretinoic acid responsive receptor at a concentration of about 0.2 μM.The hepatocyte-like cells may also be exposed to the activator of aretinoic acid responsive receptor at a concentration of about 0.5 μM.The hepatocyte-like cells may also be exposed to the activator of aretinoic acid responsive receptor at a concentration of about 0.75 μM.The hepatocyte-like cells may also be exposed to the activator of aretinoic acid responsive receptor at a concentration of about 1 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration of about 1.25 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration of about 1.5 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration of about 1.75 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration of about 2 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration of about 2.5 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration of about 3 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration of about 3.5 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration of about 4 μM. Thehepatocyte-like cells may also be exposed to the activator of a retinoicacid responsive receptor at a concentration of about 5 μM.

In case that, for instance, 9-cis-retinoic acid is employed as theactivator of a retinoic acid responsive receptor according to theinvention, it may be exposed to the hepatocyte-like cells at aconcentration in the range of about 0.1 to about 2.5 μM, such as, e.g.,in the range of about 0.1 to about 0.5 μM, such as, e.g., at about 0.2μM.

The hepatocyte-like cells may thus be exposed to 9-cis-retinoic acid ata concentration in the range of about 0.1 to about 2 μM. Thehepatocyte-like cells may also be exposed to 9-cis-retinoic acid at aconcentration in the range of about 0.1 to about 1.5 μM. Thehepatocyte-like cells may thus be exposed to 9-cis-retinoic acid at aconcentration in the range of about 0.1 to about 1 μM. Thehepatocyte-like cells may thus be exposed to 9-cis-retinoic acid at aconcentration in the range of about 0.1 to about 0.75 μM. Thehepatocyte-like cells may thus be exposed to 9-cis-retinoic acid at aconcentration in the range of about 0.1 to about 0.5 μM. Thehepatocyte-like cells may thus be exposed to 9-cis-retinoic acid at aconcentration in the range of about 0.1 to about 0.3 μM. Thehepatocyte-like cells may also be exposed to 9-cis-retinoic acid at aconcentration in the range of about 0.2 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to 9-cis-retinoic acid at aconcentration in the range of about 0.2 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to 9-cis-retinoic acid at aconcentration in the range of about 0.5 to about 2.5 μM. Thedifferentiating hepatic progenitor cells may also be exposed to9-cis-retinoic acid at a concentration in the range of about 0.5 toabout 2 μM. The hepatocyte-like cells may also be exposed to9-cis-retinoic acid at a concentration in the range of about 0.5 toabout 1.5 μM. The hepatocyte-like cells may also be exposed to9-cis-retinoic acid at a concentration in the range of about 0.5 toabout 1 μM. The hepatocyte-like cells may also be exposed to9-cis-retinoic acid at a concentration in the range of about 0.75 toabout 2.5 μM. The hepatocyte-like cells may also be exposed to9-cis-retinoic acid at a concentration in the range of about 0.75 toabout 2 μM. The hepatocyte-like cells may also be exposed to9-cis-retinoic acid at a concentration in the range of about 0.75 toabout 1.5 μM. The hepatocyte-like cells may also be exposed to9-cis-retinoic acid at a concentration in the range of about 0.75 toabout 1.25 μM. The hepatocyte-like cells may also be exposed to9-cis-retinoic acid at a concentration in the range of about 1 to about2.5 μM. The hepatocyte-like cells may also be exposed to 9-cis-retinoicacid at a concentration in the range of about 1 to about 2 μM. Thehepatocyte-like cells may also be exposed to 9-cis-retinoic acid at aconcentration in the range of about 1 to about 1.5 μM.

Thus, the hepatocyte-like cells may be exposed to 9-cis-retinoic acid ata concentration of about 0.1 μM. The hepatocyte-like cells may also beexposed to 9-cis-retinoic acid at a concentration of about 0.2 μM. Thehepatocyte-like cells may also be exposed to 9-cis-retinoic acid at aconcentration of about 0.5 μM. The differentiating hepatic progenitorcells may also be exposed to 9-cis-retinoic acid at a concentration ofabout 0.75 μM. The hepatocyte-like cells may also be exposed to9-cis-retinoic acid at a concentration of about 1 μM. Thehepatocyte-like cells may also be exposed to 9-cis-retinoic acid at aconcentration of about 1.25 μM. The hepatocyte-like cells may also beexposed to 9-cis-retinoic acid at a concentration of about 1.5 μM. Thedifferentiating hepatic progenitor cells may also be exposed to9-cis-retinoic acid at a concentration of about 1.75 μM. Thehepatocyte-like cells may also be exposed to 9-cis-retinoic acid at aconcentration of about 2 μM.

Similar concentrations may be used in case that, for instance,13-cis-retinoic acid is employed as the activator of a retinoic acidresponsive receptor according to the invention.

In addition to being exposed to the activator of a retinoic acidresponsive receptor, the hepatocyte-like cells may optionally also beexposed to a GSK-3 inhibitor or activator of Wnt signalling and/or to anoverlay of one or more components characteristic of the mammalianextracellular matrix (matrix overlay). Thus, the exposure to theactivator of a retinoic acid responsive receptor is combined with theexposure to a GSK-3 inhibitor or with the exposure to a matrix overlay,or both. The exposure to the activator of a retinoic acid responsivereceptor may also be combined with the exposure to an activator of Wntsignalling or with the exposure to a matrix overlay, or both. Theadditional exposure of the differentiating hepatic progenitor cells to aGSK-3 inhibitor or activator of Wnt signalling and/or to a matrixoverlay has shown to further improve the mature and functional featuresfor the hepatocyte-like cells (FIG. 4).

The GSK-3 inhibitor employed in the methods of the invention may be anycompound capable of inhibiting the GSK-3 signalling. Suitable GSK-3inhibitors for use in the invention are 9-Bromo-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one, also known as Kenpaullone or NSC 664704;1-Aza-Kenpaullone(9-Bromo-7,12-dihydro-pyrido[3′,2′:2,3]azepino[4,5-b]indol-6(5H)-one);Alsterpaullone(9-Nitro-7,12-dihydroindolo-[3,2-d][1]benzazepin-6(5)-one);4-(2,6-dichlorobenzamido)-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamidealso known as AT-7519;N-(5-((5-tert-butyloxazol-2-yl)methylthio)thiazol-2-yl)piperidine-4-carboxamidealso known as SNS-032 (BMS-387032);4-(1-isopropyl-2-methyl-1H-imidazol-5-yl)-N-(4-(methylsulfonyl)phenyl)pyrimidin-2-aminealso known as AZD5438; (2′Z,3′£)-6-Bromoindirubin-3′-oxime, also knownas BIO (GSK3 Inhibitor IX); (2′Z,3′E)-6-Bromoindirubin-3′-acetoxime,also known as BIO-Acetoxime (GSK3 Inhibitor X);(5-Methyl-IH-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine(GSK3-Inhibitor XIII); Pyridocarbazole-cyclopenadienylruthenium complex(GSK3 Inhibitor XV); TDZD-84-Benzyl-2-methyl-I,2,4-thiadiazolidine-3,5-dione (GSK3beta InhibitorI); 2-Thio(3-iodobenzyl)-5-(I-pyridyl)-[I,3,4]-oxadiazole (GSK3betaInhibitor II); OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione(GSK3beta Inhibitor III); alpha-4-Dibromoacetophenone (GSK3betaInhibitor VII); N-(4-Methoxybenzyl)-N′-(5-nitro-I,3-thiazol-2-yl)urea,also known as AR-AO 14418 (GSK-3beta Inhibitor VIII);3-(I-(3-Hydroxypropyl)-IH-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione(GSK-3beta Inhibitor XI); TWSI 19 pyrrolopyrimidine compound (GSK3betaInhibitor XII); L803 H-KEAPPAPPQSpP-NH2 or its myristoylated form(GSK3beta Inhibitor XIII);2-Chloro-I-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3beta Inhibitor VI);Aminopyrimidine CHIR99021;3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione,also known as SB216763; and Indirubin-3′-monoxime. The GSK-3 inhibitormay be selected from the above group.

Other suitable GSK-3 inhibitors which may be employed in the methods ofthe invention are 3F8(5-Ethyl-7,8-dimethoxy-1H-pyrrolo[3,4-c]-isoquinoline-1,3-(2H)-dione),A1070722, anorganic ions like Beryllium, Copper, Lithium, Mercury,Tungstate (Wolfram), and Zinc, AR-A 014418, AZD2858, Axin GID-25residues (peptide), bisindolylmaleimides, CHIR98014 (CT98014), CHIR98023(CT98023), FRATide-39 residues (peptide), Halomethylketone derivatives,e.g. HMK-32, KT5720, L803-mts (peptide) and variants, LY20900314, NP-12(Tideglusib, NP031112), NP00111, NP031115, Polyoxygenatedbis-7-azaindolyl-maleimides, RO31-8220, SB415286 (maleimide), TC-G24,TCS2002, TCS21311, TDZD-8, TOS119 and TWS119 (difluoroacetate). TheGSK-3 inhibitor may be selected from the above group

The GSK-3 inhibitor may, for instance, be one selected from Kenpaullone,1-Aza-Kenpaullone, Alsterpaullone, Aminopyrimidine CHIR99021 andIndirubin-3′-monoxime.

The GSK-3 inhibitor employed in the methods of the invention may beKenpaullone 9-Bromo-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one.The GSK-3 inhibitor may also be 1-Aza-Kenpaullone. The GSK-3 inhibitormay also be Alsterpaullone. The GSK-3 inhibitor may also be AT-7519. TheGSK-3 inhibitor may also be SNS-032 (BMS-387032). The GSK-3 inhibitormay also be AZD5438. The GSK-3 inhibitor may also be BIO(2′Z,3′£)-6-Bromoindirubin-3′-oxime. The GSK-3 inhibitor may also beBIO-Acetoxime (2′Z,3′E)-6-Bromoindirubin-3′-acetoxime. The GSK-3inhibitor may also be(5-Methyl-IH-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine. The GSK-3inhibitor may also be Pyridocarbazole-cyclopenadienylruthenium complex.The GSK-3 inhibitor may also be TDZD-84-Benzyl-2-methyl-I,2,4-thiadiazolidine-3,5-dione. The GSK-3 inhibitormay also be 2-Thio(3-iodobenzyl)-5-(I-pyridyl)-[I,3,4]-oxadiazole. TheGSK-3 inhibitor may also be OTDZT2,4-Dibenzyl-5-oxothiadiazolidine-3-thione. The GSK-3 inhibitor may alsobe alpha-4-Dibromoacetophenone. The GSK-3 inhibitor may also be AR-AO14418 N-(4-Methoxybenzyl)-N′-(5-nitro-I,3-thiazol-2-yl)urea. The GSK-3inhibitor may also be3-(I-(3-Hydroxypropyl)-IH-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione.The GSK-3 inhibitor may also be TWSI 19 pyrrolopyrimidine compound. TheGSK-3 inhibitor may also be L803 H-KEAPPAPPQSpP-NH2 or its myristoylatedform. The GSK-3 inhibitor may also be2-Chloro-I-(4,5-dibromo-thiophen-2-yl)-ethanone. The GSK-3 inhibitor mayalso be Aminopyrimidine CHIR99021. The GSK-3 inhibitor may also beSB2167633-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione.The GSK-3 inhibitor may also be Indirubin-3′-monoxime.

The hepatocyte-like cells may not only be exposed to one GSK-3inhibitor, but may also be exposed to one or more further GSK-3inhibitors, such as to a combination of two, three or four of thosementioned above.

The hepatocyte-like cells may generally be exposed to the GSK-3inhibitor at a concentration in the range of about 0.01 to about 10 μM.

Thus, the hepatocyte-like cells may be exposed to the GSK-3 inhibitor ata concentration in the range of about 0.05 to about 5 μM. Thehepatocyte-like cells may thus be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.05 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.05 to about 2 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.05 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.05 to about 1 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.05 to about 0.5 μM. Thehepatocyte-like cells may be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.1 to about 5 μM. Thehepatocyte-like cells may thus be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.1 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.1 to about 2 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.1 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.1 to about 1 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.1 to about 0.5 μM.

The hepatocyte-like cells may also be exposed to the GSK-3 inhibitor ata concentration in the range of about 0.25 to about 5 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.25 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.25 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.25 to about 1 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.25 to about 0.75 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.25 to about 0.5 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.5 to about 5 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.5 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.5 to about 2 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.5 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.5 to about 1 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.75 to about 5 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.75 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.75 to about 2.0 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.75 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 0.75 to about 1 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 1 to about 5 μM. The hepatocyte-likecells may also be exposed to the GSK-3 inhibitor at a concentration inthe range of about 1 to about 4 μM. The hepatocyte-like cells may alsobe exposed to the GSK-3 inhibitor at a concentration in the range ofabout 1 to about 3 μM. The hepatocyte-like cells may also be exposed tothe GSK-3 inhibitor at a concentration in the range of about 1 to about2.5 μM. The hepatocyte-like cells may also be exposed to the GSK-3inhibitor at a concentration in the range of about 1 to about 2 μM. Thehepatocyte-like cells may also be exposed to the GSK-3 inhibitor at aconcentration in the range of about 1 to about 1.5 μM.

In case that, for instance, Kenpaullone is employed as theGSK3-inhibitor, the hepatocyte-like cells may be exposed to it at aconcentration in the range of about 0.05 to about 5 μM, such as, e.g.,in the range of about 0.5 to about 1.5 μM.

The hepatocyte-like cells may be exposed to Kenpaullone at aconcentration in the range of about 0.05 to about 2 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.05 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.05 to about 1 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.05 to about 0.5 μM. Thehepatocyte-like cells may be exposed to Kenpaullone at a concentrationin the range of about 0.1 to about 2 μM. The hepatocyte-like cells mayalso be exposed to Kenpaullone at a concentration in the range of about0.1 to about 1.5 μM. The hepatocyte-like cells may also be exposed toKenpaullone at a concentration in the range of about 0.1 to about 1 μM.The hepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.1 to about 0.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.25 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.25 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.25 to about 1 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.25 to about 0.75 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.25 to about 0.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.5 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.5 to about 2 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.5 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.5 to about 1 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.75 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.75 to about 2.0 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.75 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.75 to about 1 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 1 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 1 to about 2 μM. The hepatocyte-likecells may also be exposed to Kenpaullone at a concentration in the rangeof about 1 to about 1.5 μM.

Similar concentrations may be used in case that, for instance,1-Aza-Kenpaullone or Alsterpaullone is employed as the GSK-3 inhibitoraccording to the invention.

Besides inhibiting GSK3, the GSK-3 inhibitor used according to theinvention may further exhibit inhibitory activity towards a cyclindependent kinase (CDK), such as CDK2. Examples of such dual inhibitorare Kenpaullone, AT-7519, SNS-032 (BMS-387032) and AZD5438. Furtherexamples of such dual inhibitor are 1-Aza-Kenpaullone, Alsterpaulloneand Indirubin-3′-monoxime. Yet further examples of such dual inhibitorare 2-bromo-9-nitropaullone, 2-bromo-9-trifluoromethylpaullone,2-bromopaullone, 2-iodo-9-trifluoromethylpaullone, 2-iodopaullone,2-phenyl-4-(2-thienyl)-5H-pyrido[2±3-d][1]benzazepine-6(7H)-thione,2-[2-(1-hydroxycyclohexyl)-ethinyl]-9-trifluoromethyl-paullone,2,3-dimethoxy-9-nitropaullone, 2,3-dimethoxy-9-trifluormethylpaullone,2,3-dimethoxypaullone,2-(3-hydroxy-1-propinyl)-9-trifluoromethylpaullone,2,9-dibromo-paullone,3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-propionitrile,4-methoxypaullone,4-(4-chlorophenyl)-2-(2-naphthyl)-5H-pyrido[2±3-d][1]benzazepine-6(7H)-thione,5-benzyl-9-bromopaullone, 5-iodo-indirubin.3-monoxime,5,6,7,12-tetrahydro-benzo[6±7]cyclohept[1,2-b]indole, 6 bromo indirubin,8,10-dichloropaullone, 9-bromo-2,3-dihydroxypaullone,9-bromo-2,3-dimethoxypaullone, 9-bromo-4-hydroxypaullone,9-bromo-4-methoxypaullone,9-bromo-5-ethylpaullone,9-bromo-5-(methyloxycarbonylmethyl)paullone,9-bromo-5-methylpaullone,9-bromo-5,6,7,12-tetrahydro-benzo[6±7]cyclohept[1,2-b]indole,9-bromo-5,7-bis-(tert.-butyloxycarbonyl)-paullone,9-bromo-5,7,12-tri-(tert.-butyloxycarbonyl)-paullone,9-bromo-5,12-bis-(tert.-butyloxycarbonyl)-paullone,9-bromo-7,12-dihydro-6-(hydroxyamino)-indolo[2±3-d][1]benzazepine,9-bromo-7,12-dihydro-6-methylthio-indolo[2±3-d][1]benzazepine,9-bromo-7,12-dihydro-indolo[2±3-d][1]benzazepine-6(5H)-thione,9-bromo-12-(2-hydroxyethyl)-paullone, 9-bromo-12-(2-propenyl)-paullone,9-bromo-12-ethylpaullone, 9-bromo-12-methylpaullone,9-bromo-12-methyloxycarbonyl-methylpaullone,9-bromo-12-(tert.-butyloxycarbonyl)-paullone, 9-chloropaullone,9-cyanopaullone, 9-cyano-2,3-dimethoxypaullone, 9-fluoropaullone,9-methoxypaullone, 9-methylpaullone,9-oxo-thiazolo[5,4-f]quinazoline-2-carbonitril derivatives,9-trifluoromethylpaullone, 10-bromopaullone, 11-bromopaullone,11-chloropaullone, 11-ethylpaullone, 11-methylpaullone, Aloisines(=6-phenyl[5H]pyrrolo[2,3-6]pyrazines); e.g. Aloisine A, AZD1080,bis-indole indirubin, Debromohymenialdisine, Dibromocantharelline,(E)-3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-acrylicacid methyl ester, (E)-2-(3-oxo-1-butenyl)-9-trifluoromethylpaullone,(E)-3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-acrylonitrile,Indirubin, Hymenidin, Hymenialdisine, Manzamines, e.g. Manzamine A,Meridiamins, Paullone, Pyrazolo[3,4-b]pyridine derivatives,Pryazolo[3,4-b]quinoxaline derivatives andthiazolo[5,4-f]quinazolino-9-one derivatives.

As an alternative to a GSK3 inhibitor, the hepatocyte-like cells may beexposed to an activator of Wnt signalling. The activator of Wntsignalling employed in the methods of the present invention, may be anycompound activating Wnt signalling.

Suitable activators of Wnt signalling for use in the present inventionare Wnt proteins, such as, e.g., Wnt1, Wnt2, Wnt2B/13, Wnt3A, Wnt3,Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B,Wnt10A, Wnt10B, Wnt11 and Wnt16, which may be recombinant in nature.

The activator of Wnt signalling may thus be selected from the abovegroup of Wnt proteins. The activator of Wnt signalling may, forinstance, be Wnt3A. The activator of Wnt signalling may also be Wnt5A.

The hepatocyte-like cells may generally be exposed to the activator ofWnt signalling at a concentration in the range of about 0.05 to about 10ng/ml.

Thus, the hepatocyte-like cells may be exposed to the activator of Wntsignalling at a concentration in the range of about 0.05 to about 5ng/ml. The hepatocyte-like cells may also be exposed to the activator ofWnt signalling at a concentration in the range of about 0.1 to about 5ng/ml. The hepatocyte-like cells may also be exposed to the activator ofWnt signalling at a concentration in the range of about 0.5 to about 5ng/ml. The hepatocyte-like cells may also be exposed to the activator ofWnt signalling at a concentration in the range of about 1 to about 5ng/ml. The hepatocyte-like cells may also be exposed to the activator ofWnt signalling at a concentration in the range of about 2 to about 5ng/ml. The hepatocyte-like cells may also be exposed to the activator ofWnt signalling at a concentration in the range of about 0.1 to about 2.5ng/ml. The hepatocyte-like cells may also be exposed to the activator ofWnt signalling at a concentration in the range of about 0.1 to about 2ng/ml. The hepatocyte-like cells may also be exposed to the activator ofWnt signalling at a concentration in the range of about 0.5 to about 2.5ng/ml. The hepatocyte-like cells may also be exposed to the activator ofWnt signalling at a concentration in the range of about 0.5 to about 2ng/ml. The hepatocyte-like cells may also be exposed to the activator ofWnt signalling at a concentration in the range of about 0.5 to about 1.5ng/ml. The hepatocyte-like cells may also be exposed to the activator ofWnt signalling at a concentration in the range of about 1 to about 2.5ng/ml.

In case that, for instance, Wnt3A is employed as the activator of Wntsignalling, the hepatocyte-like cells may be exposed to it at aconcentration in the range of about 0, 05 to about 10 ng/ml, such as,e.g., in the range of about 0.5 to about 2.5 μM.

Thus, the hepatocyte-like cells may be exposed to Wnt3A at aconcentration in the range of about 0.05 to about 5 ng/ml. Thehepatocyte-like cells may also be exposed to Wnt3A at a concentration inthe range of about 0.1 to about 5 ng/ml. The hepatocyte-like cells mayalso be exposed to Wnt3A at a concentration in the range of about 0.5 toabout 5 ng/ml. The hepatocyte-like cells may also be exposed to Wnt3A ata concentration in the range of about 1 to about 5 ng/ml. Thehepatocyte-like cells may also be exposed Wnt3A at a concentration inthe range of about 2 to about 5 ng/ml. The hepatocyte-like cells mayalso be exposed to Wnt3A at a concentration in the range of about 0.1 toabout 2.5 ng/ml. The hepatocyte-like cells may also be exposed to Wnt3Aat a concentration in the range of about 0.1 to about 2 ng/ml. Thehepatocyte-like cells may also be exposed to Wnt3A at a concentration inthe range of about 0.5 to about 2.5 ng/ml. The hepatocyte-like cells mayalso be exposed to Wnt3A at a concentration in the range of about 0.5 toabout 2 ng/ml. The hepatocyte-like cells may also be exposed to Wnt3A ata concentration in the range of about 0.5 to about 1.5 ng/ml. Thehepatocyte-like cells may also be exposed to Wnt3A at a concentration inthe range of about 1 to about 2.5 ng/ml.

The hepatocyte-like cells may not only be exposed to one activator ofWnt signalling, but may also be exposure to one or more furtheractivators of Wnt signalling, such as to a combination of two, three orfour of those mentioned above.

In addition to being exposed to the activator of a retinoic acidresponsive receptor, the hepatocyte-like cells may optionally also beexposed to a CDK inhibitor and/or to an overlay of one or morecomponents characteristic of the mammalian extracellular matrix (matrixoverlay). Thus, the exposure to the activator of a retinoic acidresponsive receptor is combined with the exposure to a CDK inhibitor orwith the exposure to a matrix overlay, or both. The additional exposureof the differentiating hepatic progenitor cells to a CDK inhibitorand/or to a matrix overlay has shown to further improve the mature andfunctional features for the hepatocyte-like cells (FIG. 4).

The CDK inhibitor employed in the methods of the invention may be anycompound capable of inhibiting the function (e.g., the activity) of acyclin dependent kinase (CDK). The CDK inhibitor employed in the methodsof the invention may, for instance, be an inhibitor of one or more ofCDK1, CDK2, CDK4, CDK5, CDK6, CDK7, and CDK9. The CDK inhibitor employedin the methods of the invention may be an inhibitor of cyclin dependentkinase 2 (CDK2). The CDK inhibitor employed in the methods of theinvention may, for instance, be an inhibitor of CDK1 and CDK2. The CDKinhibitor employed in the methods of the invention may, for instance, bean inhibitor of CDK2 and CDK5. The CDK inhibitor employed in the methodsof the invention may, for instance, be an inhibitor of CDK1, CDK2 andCDK5.

Suitable CDK inhibitors for use in the invention are9-Bromo-7,12-dihydro-indolo [3,2-d]-[1]benzazepin-6(5H)-one, also knownas Kenpaullone or NSC 664704;(R)-2-(6-(benzylamino)-9-isopropyl-9H-purin-2-ylamino)butan-1-ol alsoknown as Roscovitine;2-(2-chlorophenyl)-5,7-dihydroxy-8-((3S,4R)-3-hydroxy-1-methylpiperidin-4-yl)-4H-chromen-4-onealso known as Flavopiridol;4-(2,6-dichlorobenzamido)-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamidealso known as AT-7519;6-acetyl-8-cyclopentyl-5-methyl-2-(5-(piperazin-1-yl)pyridin-2-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-onehydrochloride also known as PD 0332991 HCl;N-(5-((5-tert-butyloxazol-2-yl)methylthio)thiazol-2-yl)piperidine-4-carboxamidealso known as SNS-032 (BMS-387032); JNJ-7706621;N-(6,6-dimethyl-5-(1-methylpiperidine-4-carbonyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-3-methylbutanamidealso known as PHA-793887; Dinaciclib (SCH727965);(4-butoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)(2,6-difluoro-4-methylphenyl)methanone also known as BMS-265246;N,1,4,4-tetramethyl-8-(4-(4-methylpiperazin-1-yl)phenylamino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamidealso known as PHA-848125;2-(pyridin-4-yl)-6,7-dihydro-1H-pyrrolo[3,2-c]pyridin-4(5H)-one alsoknown as PHA-767491; SCH 900776;2-(2-chlorophenyl)-5,7-dihydroxy-8-((3S,4R)-3-hydroxy-1-methylpiperidin-4-yl)-4H-chromen-4-onehydrochloride also known as Flavopiridol HCl;(4-amino-2-(1-(methylsulfonyl)piperidin-4-ylamino)pyrimidin-5-yl)(2,3-difluoro-6-methoxyphenyl)methanonealso known as R547;(2S)-1-(5-(3-methyl-1H-indazol-5-yl)pyridin-3-yloxy)-3-phenylpropan-2-aminealso known as A-674563;4-(1-isopropyl-2-methyl-1H-imidazol-5-yl)-N-(4-(methylsulfonyl)phenyl)pyrimidin-2-aminealso known as AZD5438;N5-(6-aminohexyl)-N7-benzyl-3-isopropylpyrazolo[1,5-a]pyrimidine-5,7-diaminehydrochloride also known as BS-181 HCl; CY-202; AG-024322; P276-00; ZK304709; GPC-286199; and BAY 80-3000. The CDK inhibitor may be selectedfrom the above group.

Other suitable CDK inhibitors which may be employed in the methods ofthe invention are 2-hydroxybohemine, A674563, Aminopurvanolol,BAY1000394, BMS-265246, BS-181 Butyrolactone I, CR8 S-isomer, Diaciclib(SCH727965), JNJ-7706621, N9-isopropyl-olomoucine, NU6140, NU6102,Olomoucine II, Oxindole I, P276-00, PD332991, PHA-793887, PHA-767491,PHA-848125, PNU112455A, Purvanolol A and B, R547, (R)-DRF053 andSCH900776 (MK-8776). The CDK inhibitor may be selected from the abovegroup.

The CDK inhibitor employed in the methods of the invention may, forinstance, be Kenpaullone. Kenpaullone is known to be an inhibitor ofCDK2. The CDK inhibitor may also be Roscovitine. The CDK inhibitor mayalso be Flavopiridol. The CDK inhibitor may also be AT-7519. The CDKinhibitor may also be PD 0332991 HCl. The CDK inhibitor may also beSNS-032 (BMS-387032). The CDK inhibitor may also be JNJ-7706621. The CDKinhibitor may also be PHA-793887. The CDK inhibitor may also beDinaciclib (SCH727965). The CDK inhibitor may also be BMS-265246. TheCDK inhibitor may also be PHA-848125. The CDK inhibitor may also bePHA-767491. The CDK inhibitor may also be SCH 900776. The CDK inhibitormay also be Flavopiridol HCl. The CDK inhibitor may also be R547. TheCDK inhibitor may also be A-674563. The CDK inhibitor may also beAZD5438. The CDK inhibitor may also be BS-181 HCl. The CDK inhibitor mayalso be CY-202. The CDK inhibitor may also be AG-024322. The CDKinhibitor may also be P276-00. The CDK inhibitor may also be ZK 304709.The CDK inhibitor may also be GPC-286199. The CDK inhibitor may also beBAY 80-3000.

The CDK inhibitor may be selected from the group consisting of:Kenpaullone, SNS-032(BMS-387032), AT-7519 and AZD5438.

The CDK inhibitor may be selected from the group consisting of:Kenpaullone, 1-Aza-Kenpaullone, Indirubin-3′-monoxime, Alsterpaullone,SNS-032(BMS-387032), AT-7519 and AZD5438.

The hepatocyte-like cells may not only be exposed to one CDK inhibitor,but may also be exposure to one or more further CDK inhibitors, such asto a combination of two, three or four of those mentioned above.

The hepatocyte-like cells may generally be exposed to the CDK inhibitorat a concentration in the range of about 0.01 to about 10 μM.

Thus, the hepatocyte-like cells may be exposed to the CDK inhibitor at aconcentration in the range of about 0.05 to about 5 μM. Thehepatocyte-like cells may thus be exposed to the CDK inhibitor at aconcentration in the range of about 0.05 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.05 to about 2 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.05 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.05 to about 1 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.05 to about 0.5 μM. Thehepatocyte-like cells may be exposed to the CDK inhibitor at aconcentration in the range of about 0.1 to about 5 μM. Thehepatocyte-like cells may thus be exposed to the CDK inhibitor at aconcentration in the range of about 0.1 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.1 to about 2 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.1 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.1 to about 1 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.1 to about 0.5 μM.

The hepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.25 to about 5 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.25 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.25 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.25 to about 1 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.25 to about 0.75 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.25 to about 0.5 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.5 to about 5 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.5 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.5 to about 2 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.5 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.5 to about 1 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.75 to about 5 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.75 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.75 to about 2.0 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.75 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 0.75 to about 1 μM. Thehepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 1 to about 5 μM. The hepatocyte-likecells may also be exposed to the CDK inhibitor at a concentration in therange of about 1 to about 4 μM. The hepatocyte-like cells may also beexposed to the CDK inhibitor at a concentration in the range of about 1to about 3 μM. The hepatocyte-like cells may also be exposed to the CDKinhibitor at a concentration in the range of about 1 to about 2.5 μM.The hepatocyte-like cells may also be exposed to the CDK inhibitor at aconcentration in the range of about 1 to about 2 μM. The hepatocyte-likecells may also be exposed to the CDK inhibitor at a concentration in therange of about 1 to about 1.5 μM.

In case that, for instance, Kenpaullone is employed as the CDKinhibitor, the hepatocyte-like cells may be exposed to it at aconcentration in the range of about 0.05 to about 5 μM, such as, e.g.,in the range of about 0.5 to about 1.5 μM.

The hepatocyte-like cells may be exposed to Kenpaullone at aconcentration in the range of about 0.05 to about 2 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.05 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.05 to about 1 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.05 to about 0.5 μM. Thehepatocyte-like cells may be exposed to Kenpaullone at a concentrationin the range of about 0.1 to about 2 μM. The hepatocyte-like cells mayalso be exposed to Kenpaullone at a concentration in the range of about0.1 to about 1.5 μM. The hepatocyte-like cells may also be exposed toKenpaullone at a concentration in the range of about 0.1 to about 1 μM.The hepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.1 to about 0.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.25 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.25 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.25 to about 1 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.25 to about 0.75 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.25 to about 0.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.5 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.5 to about 2 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.5 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.5 to about 1 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.75 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.75 to about 2.0 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.75 to about 1.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 0.75 to about 1 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 1 to about 2.5 μM. Thehepatocyte-like cells may also be exposed to Kenpaullone at aconcentration in the range of about 1 to about 2 μM. The hepatocyte-likecells may also be exposed to Kenpaullone at a concentration in the rangeof about 1 to about 1.5 μM.

Besides inhibiting CDK, the CDK inhibitor used according to theinvention may further exhibit inhibitory activity towards Glycogensynthase kinase 3 (GSK-3), especially GSK-3 beta. Examples of such dualinhibitor are Kenpaullone, SNS-032 (BMS-387032), AT-7519 and AZD5438which inhibit both CDK2 and GSK3. Further examples of such dualinhibitor are 1-Aza-Kenpaullone, Alsterpaullone andIndirubin-3′-monoxime. Further examples of such dual inhibitor are1-Aza-Kenpaullone, Alsterpaullone and Indirubin-3′-monoxime. Yet furtherexamples of such dual inhibitor are 2-bromo-9-nitropaullone,2-bromo-9-trifluoromethylpaullone, 2-bromopaullone,2-iodo-9-trifluoromethylpaullone, 2-iodopaullone,2-phenyl-4-(2-thienyl)-5H-pyrido[2±3-d][1]benzazepine-6(7H)-thione,2-[2-(1-hydroxycyclohexyl)-ethinyl]-9-trifluoromethyl-paullone,2,3-dimethoxy-9-nitropaullone, 2,3-dimethoxy-9-trifluormethylpaullone,2,3-dimethoxypaullone,2-(3-hydroxy-1-propinyl)-9-trifluoromethylpaullone,2,9-dibromo-paullone,3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-propionitrile,4-methoxypaullone,4-(4-chlorophenyl)-2-(2-naphthyl)-5H-pyrido[2±3-d][1]benzazepine-6(7H)-thione,5-benzyl-9-bromopaullone, 5-iodo-indirubin.3-monoxime,5,6,7,12-tetrahydro-benzo[6±7]cyclohept[1,2-b]indole, 6 bromo indirubin,8,10-dichloropaullone, 9-bromo-2,3-dihydroxypaullone,9-bromo-2,3-dimethoxypaullone, 9-bromo-4-hydroxypaullone,9-bromo-4-methoxypaullone,9-bromo-5-ethylpaullone,9-bromo-5-(methyloxycarbonylmethyl)paullone,9-bromo-5-methylpaullone,9-bromo-5,6,7,12-tetrahydro-benzo[6±7]cyclohept[1,2-b]indole,9-bromo-5,7-bis-(tert.-butyloxycarbonyl)-paullone,9-bromo-5,7,12-tri-(tert.-butyloxycarbonyl)-paullone,9-bromo-5,12-bis-(tert.-butyloxycarbonyl)-paullone,9-bromo-7,12-dihydro-6-(hydroxyamino)-indolo[2±3-d][1]benzazepine,9-bromo-7,12-dihydro-6-methylthio-indolo[2±3-d][1]benzazepine,9-bromo-7,12-dihydro-indolo[2±3-d][1]benzazepine-6(5H)-thione,9-bromo-12-(2-hydroxyethyl)-paullone, 9-bromo-12-(2-propenyl)-paullone,9-bromo-12-ethylpaullone, 9-bromo-12-methylpaullone,9-bromo-12-methyloxycarbonyl-methylpaullone,9-bromo-12-(tert.-butyloxycarbonyl)-paullone, 9-chloropaullone,9-cyanopaullone, 9-cyano-2,3-dimethoxypaullone, 9-fluoropaullone,9-methoxypaullone, 9-methylpaullone,9-oxo-thiazolo[5,4-f]quinazoline-2-carbonitril derivatives,9-trifluoromethylpaullone, 10-bromopaullone, 11-bromopaullone,11-chloropaullone, 11-ethylpaullone, 11-methylpaullone, Aloisines(=6-phenyl[5H]pyrrolo[2,3-6]pyrazines); e.g. Aloisine A, AZD1080,bis-indole indirubin, Debromohymenialdisine, Dibromocantharelline,(E)-3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-acrylicacid methyl ester, (E)-2-(3-oxo-1-butenyl)-9-trifluoromethylpaullone,(E)-3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-acrylonitrile,Indirubin, Hymenidin, Hymenialdisine, Manzamines, e.g. Manzamine A,Meridiamins, Paullone, Pyrazolo[3,4-b]pyridine derivatives,Pryazolo[3,4-b]quinoxaline derivatives andthiazolo[5,4-f]quinazolino-9-one derivatives.

It is also contemplated by the present invention that thehepatocyte-like cells being expose to a GSK3 inhibitor or activator ofWnt signalling are further exposed to a CDK inhibitor. This isparticularly of interest if the GSK inhibitor employed does not exhibitinhibitory activity towards a cyclin dependent kinase (CDK),

Likewise, is contemplated by the present invention that thehepatocyte-like cells being expose to a CDK inhibitor are furtherexposed to a GSK3 inhibitor or activator of Wnt signalling. This isparticularly of interest if the CDK inhibitor employed does not exhibitinhibitory activity towards Glycogen synthase kinase 3 (GSK-3),

Accordingly, the present invention further provides a method forpromoting the maturation of human hepatocyte-like cells whereby saidhepatocyte-like cells are exposed to an activator of a retinoic acidresponsive receptor, such as retinoic acid, optionally in combinationwith exposure to an inhibitor of GSK3 signalling or activator of Wntsignalling and a CDK inhibitor, further optionally in combination withan overlay of the cells with one or more components characteristic ofthe mammalian extracellular matrix (matrix overlay).

The method for promoting the maturation of human hepatocyte-like cellsmay thus be described as comprising the step:

Exposing said human hepatocyte-like cells to an activator of a retinoicacid responsive receptor, optionally in combination with exposure to aCDK inhibitor and a GSK3 inhibitor or activator of Wnt signalling,further optionally in combination with exposure to an overlay of thecells with one or more components characteristic of the mammalianextracellular matrix (matrix overlay).

Moreover, the present invention further provides a method for producinghuman hepatocyte-like cells whereby human hepatic progenitor cells arecultured under differentiation conditions to obtain hepatocyte-likecells, and the obtained hepatocyte-like cells are exposed to anactivator of a retinoic acid responsive receptor, such as retinoic acid,optionally in combination with exposure to an inhibitor of GSK3signalling or activator of Wnt signalling and a CDK inhibitor, furtheroptionally in combination with an overlay of the cells with one or morecomponents characteristic of the mammalian extracellular matrix (matrixoverlay).

The method for producing human hepatocyte-like cells may thus bedescribed as comprising the following steps:

Culturing human hepatic progenitor cells under differentiationconditions to obtain hepatocyte-like cells, and

Exposing said hepatocyte-like cells to an activator of a retinoic acidresponsive receptor, optionally in combination with exposure to a CDKinhibitor and a GSK3 inhibitor or activator of Wnt signalling, furtheroptionally in combination with exposure to an overlay of the cells withone or more components characteristic of the mammalian extracellularmatrix (matrix overlay).

By way of example, if hepatocyte-like cells are to be exposed to anactivator of a retinoic acid responsive receptor in combination with aGSK3 inhibitor and a CDK inhibitor, the GSK3 inhibitor may be CHIR99021and the CDK inhibitor may be Roscovitine or Flavopiridol. However, it isunderstood that any other GSK3 inhibitor or CDK inhibitor, especially ofthose specifically mentioned herein, may be employed instead.

Matrix overlays consisting of Collagen I or Matrigel (a basementmembrane mix extracted from the Engelbreth-Holm-Swarm mouse sarcoma)have been used for culturing primary hepatocytes for several decades(e.g. Dunn et al. 1991; Page et al. 2007), since it was found thatprimary hepatocytes maintain a better functionality and live longer in aso called sandwich configuration, with one extracellular matrix (ECM)layer below the cells and one ECM layer on top of the cells.Classically, Collagen I and Matrigel overlays are thick, containing e.g.125 μg Matrigel/cm² or 50 μg Collagen I/cm². However, this is notreflecting the physiological composition or thickness of the liver ECM(compare e.g. Turner et al. 2011; Wang et al. 2011).

The matrix overlay employed in the methods of the invention is a novel,more physiological combination of component present in the ECM of theadult liver and comprises, or is composed of, one or more ECMcomponents, which form part of the normal mammalian extracellular matrixenvironment. Suitable ECM components for use as matrix overlay in thepresent invention are collagen, such as collagen I, II, III, IV, V orVI, fibronectin, elastin, chondroitin sulfate proteoglycan, dermatansulfate proteoglycan, heparin proteoglycan, heparan sulfateproteoglycan, such as glypicans, syndecans or perlecans,glycosaminoglycans, nidogen/entactin, laminins, biglycan, tenascin,hyaluronans, or other ECM components, or ECM component mixturescomprising, or consisting of, e.g., collagens, laminin, fibronectin,tenascin, proteoglycans, and glycosaminoglycans.

Accordingly, the hepatocyte-like cells may be exposed to a matrixoverlay comprising, or composed of, one or more, such as two, three,four, five, six, seven, eight, nine or ten, or up to 20 of the abovementioned ECM components. Thus, the hepatocyte-like cells may be exposedto a matrix overlay comprising, or composed of, two of the abovementioned ECM components. The hepatocyte-like cells may also be exposedto a matrix overlay comprising, or composed of, three of the abovementioned ECM components. The hepatocyte-like cells may also be exposedto a matrix overlay comprising, or composed of, four of the abovementioned ECM components. The hepatocyte-like cells may also be exposedto a matrix overlay comprising, or composed of, five of the abovementioned ECM components. The hepatocyte-like cells may also be exposedto a matrix overlay comprising, or composed of, six of the abovementioned ECM components. The hepatocyte-like cells may also be exposedto a matrix overlay comprising, or composed of, seven of the abovementioned ECM components. The hepatocyte-like cells may also be exposedto a matrix overlay comprising, or composed of, eight of the abovementioned ECM components. The hepatocyte-like cells may also be exposedto a matrix overlay comprising, or composed of, nine of the abovementioned ECM components. The hepatocyte-like cells may also be exposedto a matrix overlay comprising, or composed of, ten of the abovementioned ECM components.

For example, the hepatocyte-like cells may be exposed to a matrixoverlay comprising, or composed of, collagen and fibronectin(collagen-fibronectin-matrix overlay), such as a matrix overlaycomprising, or composed of, collagen I and fibronectin (collagenI-fibronectin-matrix overlay). The hepatocyte-like cells may also beexposed to a matrix overlay comprising, or composed of, collagen, suchas collagen I, and laminin (collagen-laminin-matrix overlay). Thehepatocyte-like may also be exposed to a matrix overlay comprising, orcomposed of, collagen, such as collagen I, nidogen and laminin(collagen-nidogen-laminin-matrix overlay). The hepatocyte-like cells mayalso be exposed to a matrix overlay comprising, or composed of,collagen, such as collagen I, fibronectin and laminin(collagen-fibronectin-laminin-matrix overlay). The hepatocyte-like cellsmay also be exposed to a matrix overlay comprising, or composed of,fibronectin and laminin (fibronectin-laminin-matrix overlay). Thehepatocyte-like cells may also be exposed to a matrix overlaycomprising, or composed of, Fibronectin, nidogen and laminin(Fibronectin-nidogen-laminin-matrix overlay).

The hepatocyte-like cells may also be exposed to a matrix overlaycomprising, or composed of, fibronectin, collagen I, collagen IV,collagen VI, nidogen, biglycan and laminin (fibronectin-collagen I, IV,VI-Nidogen-biglycan-laminin-matrix overlay).

The hepatocyte-like cells may also be exposed to a matrix overlaycomprising, or composed of, fibronectin, collagen I, collagen IV,nidogen, biglycan and laminin (fibronectin-collagen I,IV-nidogen-biglycan-laminin-matrix overlay).

The hepatocyte-like cells may also be exposed to a matrix overlaycomprising collagen, such as collagen I, laminin, fibronectin,proteoglycans and glycosaminoglycans (collagen, laminin, fibronectin,proteoglycans and glycosaminoglycans matrix overlay). Thehepatocyte-like cells may also be exposed to a matrix overlay comprisingcollagen, such as collagen I, laminin, fibronectin, and proteoglycans(collagen, laminin, fibronectin, and proteoglycans matrix overlay).

The hepatocyte-like cells may also be exposed to a matrix overlaycomprising collagen, such as collagen I, laminin, fibronectin, tenascin,elastin, proteoglycans and glycosaminoglycans (collagen, laminin,fibronectin, tenascin, elastin, proteoglycans and glycosaminoglycansmatrix overlay).

The matrix overlay employed in the methods of the invention is thincompared to the thick matrices so far used. The thickness of the matrixoverlay thereby correlates with the concentration of the ECM componentsemployed. Suitable concentrations for the present matrix overlay aree.g. 0.01-30 μg, such as 0.01-20 μg, ECM component/cm².

Thus, each ECM component may be present in the matrix overlay at aconcentration from about 0.1 to about 20 μg ECM component/cm². Each ECMcomponent may also be present in the matrix overlay at a concentrationfrom about 0.1 to about 15 μg ECM component/cm². Each ECM component mayalso be present in the matrix overlay at a concentration from about 0.1to about 12.5 μg protein component/cm². Each ECM component may also bepresent in the matrix overlay at a concentration from about 0.1 to about10 μg ECM component/cm². Each ECM component may also be present in thematrix overlay at a concentration from about 0.1 to about 5 μg ECMcomponent/cm². Each ECM component may also be present in the matrixoverlay at a concentration from about 0.1 to about 2.5 μg ECMcomponent/cm². Each ECM component may also be present in the matrixoverlay at a concentration from about 0.2 to about 20 μg ECMcomponent/cm². Each ECM component may also be present in the matrixoverlay at a concentration from about 0.2 to about 15 μg ECMcomponent/cm². Each ECM component may also be present in the matrixoverlay at a concentration from about 0.2 to about 12.5 μg ECMcomponent/cm². Each ECM component may also be present in the matrixoverlay at a concentration from about 0.2 to about 10 μg ECMcomponent/cm². Each ECM component may also be present in the matrixoverlay at a concentration from about 0.2 to about 5 μg ECMcomponent/cm². Each ECM component may also be present in the matrixoverlay at a concentration from about 0.5 to about 25 μg ECMcomponent/cm². Each ECM component may also be present in the matrixoverlay at a concentration from about 0.5 to about 20 μg ECMcomponent/cm². Each protein component may also be present in the matrixoverlay at a concentration from about 0.5 to about 15 μg proteincomponent/cm². Each protein component may also be present in the matrixoverlay at a concentration from about 0.5 to about 10 μg proteincomponent/cm². Each protein component may also be present in the matrixoverlay at a concentration from about 0.5 to about 7.5 μg proteincomponent/cm². Each protein component may also be present in the matrixoverlay at a concentration from about 0.5 to about 5 μg proteincomponent/cm². Each protein component may also be present in the matrixoverlay at a concentration from about 0.5 to about 2.5 μg proteincomponent/cm². Each protein component may also be present in the matrixoverlay at a concentration from about 0.5 to about 1.5 μg proteincomponent/cm². Each protein component may also be present in the matrixoverlay at a concentration from about 0.5 to about 1 μg proteincomponent/cm². Each ECM component may be present in the matrix overlayat a concentration from about 1 to about 20 μg ECM component/cm². EachECM component may also be present in the matrix overlay at aconcentration from about 1 to about 15 μg ECM component/cm². Each ECMcomponent may also be present in the matrix overlay at a concentrationfrom about 1 to about 12.5 μg protein component/cm². Each ECM componentmay also be present in the matrix overlay at a concentration from about1 to about 10 μg ECM component/cm². Each ECM component may also bepresent in the matrix overlay at a concentration from about 1 to about 5μg ECM component/cm². Each ECM component may also be present in thematrix overlay at a concentration from about 1 to about 2.5 μg ECMcomponent/cm². Each ECM component may also be present in the matrixoverlay at a concentration from about 2 to about 20 μg ECMcomponent/cm². Each ECM component may also be present in the matrixoverlay at a concentration from about 2 to about 15 μg ECMcomponent/cm². Each ECM component may also be present in the matrixoverlay at a concentration from about 2 to about 12.5 μg ECMcomponent/cm². Each ECM component may also be present in the matrixoverlay at a concentration from about 2 to about 10 μg ECMcomponent/cm². Each ECM component may also be present in the matrixoverlay at a concentration from about 2 to about 5 μg ECM component/cm².Each ECM component may also be present in the matrix overlay at aconcentration from about 5 to about 25 μg ECM component/cm². Each ECMcomponent may also be present in the matrix overlay at a concentrationfrom about 5 to about 20 μg ECM component/cm². Each protein componentmay also be present in the matrix overlay at a concentration from about5 to about 15 μg protein component/cm². Each protein component may alsobe present in the matrix overlay at a concentration from about 5 toabout 10 μg protein component/cm². Each protein component may also bepresent in the matrix overlay at a concentration from about 5 to about7.5 μg protein component/cm².

Collagen I may, for example, be present in the matrix overlay at aconcentration from about 2 to about 20 μg/cm², such as from about 5 toabout 15 μg/cm².

Collagen IV may, for example, be present in the matrix overlay at aconcentration from about 0.01 to 10 μg/cm², such as from about 0.05 toabout 10 μg/cm².

Collagen VI may, for example, be present in the matrix overlay at aconcentration form about 0.01 to about 15 μg/cm², such as from about0.01 to about 10 μg/cm² or from about 0.1 to about 15 μg/cm².

Fibronectin may, for example, be present in the matrix overlay at aconcentration from about 2 to about 30 μg/cm², such as from about 2 toabout 20 μg/cm².

Nidogen may, for example, be present in the matrix overlay at aconcentration from about 0.01 to about 10 μg/cm², such as from about0.05 to about 10 μg/cm².

Laminin may, for example, be present in the matrix overlay at aconcentration from about 0.01 to about 10 μg/cm², such as from about0.05 to about 10 μg/cm².

Biglycan may, for example, be present in the matrix overlay at aconcentration from about 0.01 to about 10 μg/cm², such as from about 0.1to about 10 μg/cm².

It is to be understood that the above concentrations in “μg/cm²” arewith respect to the respective component in its dry state.

Generally, cells may be cultured on a coating as growth support whichcovers the surface of the culture vessel. Gelatine or fibronectin basedcoating are widely used as growth support. Thus, the cells, inparticular the hepatic progenitor cells and hepatocyte-like cells, maybe cultured on a gelatin or fibronectin based coating. However, thecells may also be cultured on a coating which has a composition similaror identical to a matrix overlay as defined above. For example, when amatrix overlay is to be employed, the cells may be cultured on a coatingwhich has a composition which is identical to that of the employedmatrix overlay. Accordingly, a so-called “sandwich” type cultureenvironment is provided.

As an optional pre-step, the hepatic progenitor cells used in themethods of the invention may initially be derived from human pluripotentstem (hPS) cells, such as human embryonic stem (hES) cells or humaninduced pluripotent stem cells (hiPS). The methods of the invention maythus further comprise as an initial step the culturing of hPS cellsunder differentiation conditions to obtain said hepatic progenitorcells. Thus, hPS cells are initially differentiated into said hepaticprogenitor cells. This step is referred to herein as initial hepaticdifferentiation.

As indicated above, the human pluripotent stem cells which may also beused as starting material to obtain the endodermal and/or hepaticprogenitor cells may be human embryonic stem cells. Various techniquesfor obtaining such hES cells are known to the skilled person.Preferably, however, the hES cells for use according to the inventionare ones which have been derived (or obtained) without destruction ofthe human embryo, such as by employing the single blastomere removaltechnique described in e.g. Chung et al (2008), further described byMercader et al. in Essential Stem Cell Methods (First Edition, 2009).Suitable hES cell lines for use are, for example, the cell lines SA167,SA181, SA461 (Cellartis AB, Göteborg, Sweden) which are listed in theNIH stem cell registry, the UK Stem Cell bank and the European hESCregistry and are available on request. Other suitable cell lines for useare those established by Klimanskaya et al. (2006), such as cell linesMA01 and MA09, and Chung et al. (2008), such as cell lines MA126, MA127,MA128 and MA129, which all are listed with the International Stem CellRegistry (assigned to Advanced Cell Technology, Inc. Worcester, Mass.,USA).

Alternatively, the human pluripotent stem cells which may be used asstarting material to obtain the endodermal and/or hepatic progenitorcells may be human induced pluripotent stem cells. Various techniquesfor obtaining such hiPS cells have been described in the scientificliterature, and are thus known to the skilled person [see, e.g.,Takahashi et al. (2007); Zhou et al. (2009); Yu and Thomson inEssentials of Stem Cell Biology (2^(nd) Edition].

It is also envisaged that the endodermal and/or hepatic progenitor cellsmay also be derived from other pluripotent stem cells such as adult stemcells, cancer stem cells or from other embryonic, fetal, juvenile oradult sources.

Suitable conditions for differentiating hPS cells into hepaticprogenitor cells are known (see, e.g., Hay 2008, Brolen 2010 and Duan2010). WO 2009/013254 A1, for example, describes suitable protocols toobtain hepatic progenitor cells (Embodiments 1 to 4).

The hPS cells are normally cultured for up to 14 days in suitabledifferentiation medium in order to obtain hepatic progenitor cells. Forexample, the hPS cells may be cultured in suitable differentiationmedium for about 10 to about 14 days, such as for about 11 to 14 days.

The initial hepatic differentiation may be defined by including apre-endodermal step, i.e. the culturing of the hPS cells underdifferentiation conditions to obtain cells of the definitive endoderm(DE cells), which is followed by a pre-hepatic step, i.e. the culturingof the obtained DE cells under differentiation conditions to obtain thehepatic progenitor cells. Accordingly, hPS cells are firstdifferentiated into definitive endoderm, followed by the furtherdifferentiation of the definitive endoderm into hepatic progenitorcells.

Generally, in order to obtain endodermal cells, hPS cells are culturedin a differentiation medium comprising activin, such as activin A or B.The differentiation medium may further include a histone deacetylase(HDAC) inhibitor, such as Sodium Butyrate (NaB), Phenylbutyrate (PB),valproate, trichostatin A, Entinostat or Panobinstat. Thedifferentiation medium may further comprise one or more growth factors,such as FGF1, FGF2 and FGF4, and/or serum, such as FBS or FCS. Thedifferentiation medium may comprise a GSK3-inhibitor, such as, e.g.,CHIR99021, or an activator of Wnt signalling, such as Wnt3A. Thedifferentiation medium may further comprise a PI3K (Phosphoinositide3-kinase) inhibitor, such as LY294002.

The concentration of activin is usually in the range of about 50 toabout 150 ng/ml, such as about 80 to about 120 ng/ml. Activin may, forexample, be present in the differentiation medium at a concentration ofabout 50 ng/ml or about 100 ng/ml. The concentration of the HDACinhibitor is usually in the range of about 0.5 to about 2 mM. The HDACinhibitor may, for example, be present in the differentiation medium ata concentration of about 0.5 mM or about 1 mM. The concentration of theone or more growth factors may vary depending on the particular compoundused. The concentration of FGF2, for example, is usually in the range ofabout 2 to about 50 ng/ml, such as about 2 to about 10 ng/ml. FGF2 may,for example, be present in the differentiation medium at a concentrationof about 4 or about 5 ng/ml. The concentration of FGF1, for example, isusually in the range of about 50 to about 200 ng/ml, such as about 80 toabout 120 ng/ml. FGF1 may, for example, be present in thedifferentiation medium at a concentration of about 100 ng/ml. Theconcentration of FGF4, for example, is usually in the range of about 20to about 40 ng/ml. FGF4 may, for example, be present in thedifferentiation medium at a concentration of about 30 ng/ml. Theconcentration of serum, if present, is usually in the range of about 0.1to about 2% v/v, such as about 0.1 to about 0.5%, about 0.2 to about1.5% v/v, about 0.2 to about 1% v/v, about 0.5 to 1% v/v or about 0.5 toabout 1.5% v/v. Serum may, for example, be present in thedifferentiation medium at a concentration of about 0.2% v/v, about 0.5%v/v or about 1% v/v. The concentration of the GSK3 inhibitor, ifpresent, is usually in the range of about 0.1 to about 10 μM, such asabout 0.05 to about 5 μM. The concentration of the activator of Wntsignalling, if present, is usually in the range of about 0.05 to about10 ng/ml, such as about 0, 5 to about 5 μM. The concentration of thePI3K inhibitor, for example, is usually in the range of about 0.1 to 10μM, such as about 1 to 5 μM.

The differentiation medium may further comprise other supplements suchas PEST and/or GlutaMAX. The differentiation medium may also furthercomprise a ROCK inhibitor. The concentration of PEST is usually in therange of about 0.1 to about 0.5% v/v, such as about 0.1 to about 0.25%v/v. The concentration of GlutaMAX is usually in the range of about 0.5to about 1.5% v/v, such as about 0.75 to 1.25% v/v, e.g. about 1% v/v.The differentiation medium may also further comprise a ROCK inhibitor.The concentration of the ROCK inhibitor is usually in the range of about1 to about 10 μM, such as about 2.5 to about 7.5 μM, e.g., about 5 μM.

The culture medium forming the basis for the differentiation medium maybe any culture medium suitable for culturing hPS cells such as RPMI 1640or advanced medium, Dulbecco's Modified Eagle Medium (DMEM), HCM medium,HBM medium or Williams E based medium. Thus, the differentiation mediummay be RPMI 1640 or advanced medium comprising or supplemented with theabove-mentioned components. Alternatively, the differentiation mediummay be DMEM comprising or supplemented with the above-mentionedcomponents. The differentiation medium may thus also be HCM mediumcomprising or supplemented with the above-mentioned components. Thedifferentiation medium may thus also be HBM medium comprising orsupplemented with the above-mentioned components. The differentiationmedium may thus also be Williams E based medium comprising orsupplemented with the above-mentioned components.

For endodermal differentiation, hPS cells are normally cultured for upto 10 days in an activin containing differentiation medium as describedabove. The hPS cells may, for example, be cultured in saiddifferentiation medium for about 4 to about 10 days, such as for about 7to about 9 days.

Thereafter, the obtained DE cells are further cultured in adifferentiation medium comprising DMSO to obtain hepatic progenitorcells. Alternatively, the obtained DE cells may be cultured in adifferentiation medium comprising one or more growth factors, such asFGF1, FGF2 and FGF4, and one or more bone morphogenic proteins, such asBMP2 and BMP4. The differentiation medium may further comprise HGF, EGFand/or serum.

The concentration of DMSO is usually in the range of about 0.1% to about2% v/v, such as about 0.5% to about 1.5% v/v. DMSO may, for example, bepresent in the differentiation medium at a concentration of about 1%.The concentration of the one or more growth factors may vary dependingon the particular compound used. The concentration of FGF2, for example,is usually in the range of about 2 to about 50 ng/ml, such as about 2 toabout 10 ng/ml. FGF2 may, for example, be present in the differentiationmedium at a concentration of 4 or 5 ng/ml. The concentration of FGF1,for example, is usually in the range of about 50 to about 200 ng/ml,such as about 80 to about 120 ng/ml. FGF1 may, for example, be presentin the differentiation medium at a concentration of about 100 ng/ml. Theconcentration of FGF4, for example, is usually in the range of about 20to about 40 ng/ml. FGF4 may, for example, be present in thedifferentiation medium at a concentration of about 30 ng/ml. Theconcentration of HGF, if present, is usually in the range of about 10 toabout 30 ng/ml. HGF may, for example, be present in the differentiationmedium at a concentration of about 20 ng/ml. The concentration of EGF,if present is usually in the range of about 5 to about 15 ng/ml. EGFmay, for example, be present in the differentiation medium at aconcentration of about 10 ng/ml. The concentration of serum, if present,is usually in the range of about 0.1 to about 2% v/v, such as such asabout 0.1 to about 0.5%, about 0.2 to about 1.5% v/v, about 0.2 to about1% v/v, about 0.5 to 1% v/v or about 0.5 to about 1.5% v/v. Serum may,for example, be present in the differentiation medium at a concentrationof about 0.2% v/v, about 0.5% v/v or about 1% v/v.

The differentiation medium may further comprise other supplements suchas PEST and/or GlutaMAX. The concentration of PEST is usually in therange of about 0.1 to about 0.5% v/v, such as about 0.1 to about 0.25%v/v. The concentration of GlutaMAX is usually in the range of about 0.5to about 1.5% v/v, such as about 0.75 to 1.25% v/v, e.g. about 1% v/v.

The culture medium forming the basis for the differentiation medium maybe any culture medium suitable for culturing human endodermal cells suchas RPMI 1640 or advanced medium, Dulbecco's Modified Eagle Medium(DMEM), HCM medium, HBM medium or Williams E based medium. Thus, thedifferentiation medium may be RPMI 1640 or advanced medium comprising orsupplemented with the above-mentioned components. Alternatively, thedifferentiation medium may be DMEM comprising or supplemented with theabove-mentioned components. The differentiation medium may thus also beHCM medium comprising or supplemented with the above-mentionedcomponents. The differentiation medium may thus also be HBM mediumcomprising or supplemented with the above-mentioned components. Thedifferentiation medium may thus also be Williams E based mediumcomprising or supplemented with the above-mentioned components.

For differentiation into hepatic progenitor cells, DE cells are normallycultured for up to 7 days in differentiation medium as described above.The DE cells may, for example, be cultured in differentiation medium forabout 4 to about 7 days.

Basic, non-limiting culture conditions for obtaining DE cells, hepaticprogenitor cells and hepatocyte-like cells are provided in Example 2herein.

The differentiating hPS cells may also be exposed to a DNA demethylatingagent. Cells may be exposed to (or treated with) said agent at any stagebetween pluripotent stem cell stage and definitive endodermal stage.Thus, the exposure to said DNA demethylating agent may take place duringthe differentiation of the hPS cells into DE cells, i.e. during thepre-endodermal step. The cells are then cultured through endodermalstage until hepatic progenitor stage is reached, i.e. until hepaticprogenitor cells are obtained, at which point the furtherdifferentiation and maturation into hepatocyte-like cells including theexposure to the activator of a retinoic acid responsive receptor, eitheralone or in combination with GSK-3 inhibition or activation of Wntsignalling and/or a CDK inhibitor and/or a matrix overlay, is carriedout.

The DNA demethylating agent employed in the methods according to theinvention may be any compound that interferes with DNA methyltransferaseenzyme activity. Suitable DNA demethylating agents are ones of thenucleoside-analog type, such as cytidine analogues, e.g.5-aza-2-deoxycytidine (decitabine), 5-azacytidine (azacitidine) orzebularine, and of the non-nucleoside type, such as procaine, RG108,S-5-adenosyl-L-homocysteine, Caffeic acid, Chlorogenic acid,Epogallocatechin gallate, Hydralazine hydrochloride, Procainamidehydrochloride or Psammaplin A.

Thus, the DNA demethylating agent employed in the methods of theinvention may be one of the nucleoside-analogue type. Alternatively, theDNA demethylating agent employed in the methods of the invention may beone of the non-nucleoside type.

Accordingly, the DNA demethylating agent employed in the methods of theinvention may be a cytidine analogue, such as e.g. 5-aza-2-deoxycytidine(decitabine), 5-azacytidine (azacitidine), zebularine,Pseudoisocytidine, 5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine,2′-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine,2′,2′-Difluoro-deoxycytidine (gemcitabine), orCytosine-beta-D-arabinofurasonide.

The DNA demethylating agent employed in the methods of the invention maythus be a cytidine analogue selected from the group consisting of5-aza-2-deoxycytidine (decitabine), 5-azacytidine (azacitidine),5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine,2′-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine and2′,2′-Difluoro-deoxycytidine (gemcitabine).

The DNA demethylating agent employed in the methods of the invention maythus be a cytidine analogue selected from the group consisting of5-aza-2-deoxycytidine (decitabine) and 5-azacytidine (azacitidine),Alternatively, the DNA demethylating agent employed in the methods ofthe invention may be a cytidine analogue which is not5-aza-2-deoxycytidine (decitabine) or 5-azacytidine (azacitidine).

Accordingly, the DNA demethylating agent employed in the methods of theinvention may be 5-aza-2-deoxycytidine. The DNA demethylating agent mayalso be 5-azacytidine. The DNA demethylating agent may also bezebularine. The DNA demethylating agent may also be Pseudoisocytidine.The DNA demethylating agent may also be 5-fluoro-2-deoxycytidine. TheDNA demethylating agent may also be 5,6-dihydro-5-azacytidine. The DNAdemethylating agent may also be 2′-deoxy-5,6-dihydro-5-azacytidine. TheDNA demethylating agent may also be 6-azacytidine. The DNA demethylatingagent may also be 2′,2′-Difluoro-deoxycytidine (gemcitabine). The DNAdemethylating agent may also be Cytosine-beta-D-arabinofurasonide. TheDNA demethylating agent may also be procaine. The DNA demethylatingagent may also be RG108. The DNA demethylating agent may also beS-5-adenosyl-L-homocysteine. The DNA demethylating agent may also beCaffeic acid. The DNA demethylating agent may also be Chlorogenic acid.The DNA demethylating agent may also be Epogallocatechin gallate. TheDNA demethylating agent may also be Hydralazine hydrochloride. The DNAdemethylating agent may also be Procainamide hydrochloride. The DNAdemethylating agent may also be Psammaplin A.

The differentiating hPS cells may not only be exposed to one DNAdemethylating agent, but may also be exposure to one or more further DNAdemethylating agents, such as to a combination of two, three or four ofthose mentioned above.

The differentiating hPS cells may generally be exposed to the DNAdemethylating agent at a concentration in the range of about 1 nM toabout 10 μM, such as in the range of about 1 nM to about 5 μM.

Thus, the differentiating hPS cells may be exposed to the DNAdemethylating agent at a concentration in the range of about 1 nM toabout 1 μM. The differentiating hPS cells may thus be exposed to the DNAdemethylating agent at a concentration in the range of about 1 nM toabout 500 nM. The differentiating hPS cells may also be exposed to theDNA demethylating agent at a concentration in the range of about 1 nM toabout 250 nM. The differentiating hPS cells may also be exposed to theDNA demethylating agent at a concentration in the range of about 1 nM toabout 100 nM. The differentiating hPS cells may thus be exposed to theDNA demethylating agent at a concentration in the range of about 1 nM toabout 50 nM. The differentiating hPS cells may thus be exposed to theDNA demethylating agent at a concentration in the range of about 1 nM toabout 25 nM. The differentiating hPS cells may thus be exposed to theDNA demethylating agent at a concentration in the range of about 1 nM toabout 15 nM. The differentiating hPS cells may also be exposed to theDNA demethylating agent at a concentration in the range of about 1 nM toabout 10 nM. The differentiating hPS cells may also be exposed to theDNA demethylating agent at a concentration in the range of about 5 nM toabout 500 nM. The differentiating hPS cells may thus be exposed to theDNA demethylating agent at a concentration in the range of about 5 nM toabout 250 nM. The differentiating hPS cells may also be exposed to theDNA demethylating agent at a concentration in the range of about 5 nM toabout 100 nM. The differentiating hPS cells may also be exposed to theDNA demethylating agent at a concentration in the range of about 5 nM toabout 50 nM. The differentiating hPS cells may also be exposed to theDNA demethylating agent at a concentration in the range of about 5 nM toabout 25 nM. The differentiating hPS cells may also be exposed to theDNA demethylating agent at a concentration in the range of about 5 nM toabout 15 nM, such as in the range of about 10 nM. The differentiatinghPS cells may also be exposed to the DNA demethylating agent at aconcentration in the range of about 7.5 nM to about 250 nM. Thedifferentiating hPS cells may also be exposed to the DNA demethylatingagent at a concentration in the range of about 7.5 nM to about 100 nM.The differentiating hPS cells may also be exposed to the DNAdemethylating agent at a concentration in the range of about 7.5 nM toabout 50 nM. The differentiating hPS cells may also be exposed to theDNA demethylating agent at a concentration in the range of about 7.5 nMto about 25 nM. The differentiating hPS cells may also be exposed to theDNA demethylating agent at a concentration in the range of about 7.5 nMto about 12.5 nM.

In case that, for instance, 5-aza-2-deoxycytidine is employed as the DNAdemethylating agent, the differentiating hPS cells may be exposed to itat a concentration in the range of 1 nM to about 1 μM. Thedifferentiating hPS cells may thus be exposed to 5-aza-2-deoxycytidineat a concentration in the range of about 1 nM to about 500 nM. Thedifferentiating hPS cells may also be exposed to 5-aza-2-deoxycytidineat a concentration in the range of about 1 nM to about 250 nM. Thedifferentiating hPS cells may also be exposed to 5-aza-2-deoxycytidineat a concentration in the range of about 1 nM to about 100 nM. Thedifferentiating hPS cells may thus be exposed to 5-aza-2-deoxycytidineat a concentration in the range of about 1 nM to about 50 nM. Thedifferentiating hPS cells may thus be exposed to 5-aza-2-deoxycytidineat a concentration in the range of about 1 nM to about 25 nM. Thedifferentiating hPS cells may thus be exposed to 5-aza-2-deoxycytidineat a concentration in the range of about 1 nM to about 15 nM. Thedifferentiating hPS cells may also be exposed to 5-aza-2-deoxycytidineat a concentration in the range of about 1 nM to about 10 nM. Thedifferentiating hPS cells may also be exposed to 5-aza-2-deoxycytidineat a concentration in the range of about 5 nM to about 500 nM. Thedifferentiating hPS cells may thus be exposed to 5-aza-2-deoxycytidineat a concentration in the range of about 5 nM to about 250 nM. Thedifferentiating hPS cells may also be exposed to 5-aza-2-deoxycytidineat a concentration in the range of about 5 nM to about 100 nM. Thedifferentiating hPS cells may also be exposed to 5-aza-2-deoxycytidineat a concentration in the range of about 5 nM to about 50 nM. Thedifferentiating hPS cells may also be exposed to 5-aza-2-deoxycytidineat a concentration in the range of about 5 nM to about 25 nM. Thedifferentiating hPS cells may also be exposed to 5-aza-2-deoxycytidineat a concentration in the range of about 5 nM to about 15 nM, such as inthe range of about 10 nM. The differentiating hPS cells may also beexposed to 5-aza-2-deoxycytidine at a concentration in the range ofabout 7.5 nM to about 250 nM. The differentiating hPS cells may also beexposed to 5-aza-2-deoxycytidine at a concentration in the range ofabout 7.5 nM to about 100 nM. The differentiating hPS cells may also beexposed to 5-aza-2-deoxycytidine at a concentration in the range ofabout 7.5 nM to about 50 nM. The differentiating hPS cells may also beexposed to 5-aza-2-deoxycytidine at a concentration in the range ofabout 7.5 nM to about 25 nM. The differentiating hPS cells may also beexposed to 5-aza-2-deoxycytidine at a concentration in the range ofabout 7.5 nM to about 12.5 nM.

Similar concentrations may be used in case that 5-azacytidine orzebularine are employed as the DNA demethylating agent. Similarconcentrations may also be used in case of other cytidine analogues,such as, e.g. Pseudoisocytidine, 5-fluoro-2-deoxycytidine,5,6-dihydro-5-azacytidine, 2′-deoxy-5,6-dihydro-5-azacytidine,6-azacytidine, 2′,2′-Difluoro-deoxycytidine (gemcitabine), orCytosine-beta-D-arabinofurasonide, in particular5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine,2′-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine or2′,2′-Difluoro-deoxycytidine (gemcitabine).

The differentiating hPS cells are usually exposed to the DNAdemethylating agent of the nucleoside-analog type (e.g.5aza-2deoxycytidine, 5-azacytidine, zebularine) when they show greatestproliferative capacity as evidenced by cell doubling time, such asbetween day 2 and 7 of differentiation. Thus, the DNA demethylatingagent may be added to the differentiation medium on day 2 ofdifferentiation. The DNA demethylating agent may also be added to thedifferentiation medium on day 3 of differentiation. The DNAdemethylating agent may also be added to the differentiation medium onday 4 of differentiation. The DNA demethylating agent may also be addedto the differentiation medium on day 5 of differentiation. The DNAdemethylating agent may also be added to the differentiation medium onday 6 of differentiation. DNA demethylation agents of the non-nucleosidetype (e.g. procaine, RG108, S-5-adenosyl-L-homocysteine) can be added atany time in the differentiation protocol since they do not require cellproliferation to have an effect.

As shown in FIG. 13 (Example 10) herein, treatment of differentiatinghPS cells with a DNA demethylating agent surprisingly leads to animproved morphology and yield of DE cells. Moreover, treatment with aDNA demethylating agent also surprisingly led to a significantdown-regulation of expression of the stem cell marker Oct4 in DE cells(FIGS. 13 C and D) and to an improved expression of DE specific markersSOX17, CXCR4 and HHEX (FIG. 13 D). This aspect of the invention isbelieved to be the first time that a specific synergistic effect hasbeen shown between DNA demethylation and the application of specificgrowth factors whose action at a genomic level may be enhanced by thewidespread absence of methylation. Moreover, a strong synergistic effecton the maturation of hepatocyte-like cells is seen when treating cellswith a DNA demethylating agent during early endodermal developmentbefore exposing the obtained hepatic progenitor cells to an activator ofa retinoic acid responsive receptor, a GSK3 inhibitor/activator of Wntsignalling or CDK inhibitor and/or a matrix overlay (Example 8; FIGS. 9,10, and 12).

Further, the hepatocyte-like cells of the present invention may beobtained under xeno-free conditions. As such, the starting materialemployed in the methods of the invention may thus be xeno-free, such asxeno-free hPS cells or cell lines, or xeno-free hepatic progenitor cellsor cell lines which have been obtained or established under animal-freeconditions. Moreover, throughout the methods of the invention cells maybe cultured completely under xeno-free conditions, giving rise to trulyxeno-free hepatocyte-like cells. Such cells or cell line would be bettersuited to therapeutic or regenerative medicine applications and could bedistinguished from a non-xeno free composition by the presence innon-xeno free cells of the non-human sialic acid Neu5Gc or othernon-human markers (Martin et al 2005).

As a result of the methods of the present invention, hepatocyte-likecells obtained with more mature and functional features compared tocurrently available state of the art methods.

The hepatocyte-like cell(s) obtained by employing the methods of theinvention show elevated expression of hepatocyte-associated genes suchas e.g. CYP1A2, CYP3A4, CYP2C9, CYP7A1, CYP2B6, CYP3A5, MRP2, CAR, NTCP,GSTA-1, PXR and adult isoforms of HNF4a. They further show increasedmetabolic activity, as evidenced by increased activity of CYP enzymessuch as CYP1A1/2, 2C9 and 3A4/5/7 to metabolise drugs such asparacetamol and Diclofenac or UGT enzymes (UDP-glucuronyltransferases)such as UGT1A1, UGT1A9 and UGT2B7. The hepatocyte-like cell (s) of theinvention show also a prolonged longevity compared to untreated controlcells. The hepatocyte-like cell(s) obtained by employing the methods ofthe invention also show cytochrome P450 activities exceeding a foldchange of at least 1.5, such as e.g. 2, when compared to cultures where5aza-deoxycytodine, retinoic acid, Kenpaullone and a matrix overlay isnot used.

Moreover, the obtained populations of hepatocyte-like cells or cellcompositions are more pure and homogenous compared to ones obtained byfollowing currently available state of the art methods.

The cell composition(s) of the invention may further be characterized inthat at least 70% such as e.g. 75%, 80%, 90% or 95% of the cells arehepatocyte-like cells of the present invention.

The hepatocyte-like cells obtained by employing the methods of theinvention and principles as laid out in present invention may be used toa multitude of purposes comprising drug discovery processes, toxicitytest, for studying drug transporters, drug metabolizing enzyme, as invitro models for studying hepatogenesis, such as, e.g., earlyhepatogenesis, for studying human hepatoregenerative disorders, for invitro hepatotoxicity testing.

Further the hepatocyte-like cells obtained by employing the methods ofthe invention may be used for therapeutic purposes comprising: in amedicament, for the manufacture of a medicament or medicinal product forthe prevention and/or treatment of pathologies and/or diseases caused bytissue degeneration, such as, e.g., the degeneration of liver tissue.The hepatocyte-like cells of the present invention may also be used forthe manufacture of a medicament or medicinal product for the treatmentof liver disorders. Liver disorders are, for example, auto immunedisorders including primary biliary cirrhosis; metabolic disordersincluding dyslipidemia; liver disorders caused by e.g. alcohol abuse;diseases caused by viruses such as, e.g., hepatitis B, hepatitis C, andhepatitis A; liver necrosis caused by acute toxic reactions to e. g.pharmaceutical drugs; and tumour removal in patients suffering from e.g. hepatocellular carcinoma.

Alternatively, the hepatocyte-like cells obtained by employing themethods of the invention may be used for the manufacture of a medicamentor medicinal product for the treatment and/or prevention of metabolicpathologies and/or diseases.

The medicament or medicinal product may, for example, be in the form ofa replacement tissue or cell injection.

The differentiation and maturation of hepatocyte-like cells inaccordance to the invention may be useful for obtaining metabolicallyimproved hepatocyte-like cells, for studying maturation towardshepatocyte-like cells or for screening a compound for its ability tomodulate hepatocellular function, comprising exposing in vitro derivedhepatocyte-like cells obtained according to the directions providedherein to the compound, determining any phenotypic or metabolic changesin the cells that result from contact with the compound, and correlatingthe change with an ability to modulate hepatocellular function.

The invention also provides kits. Such kits are particularly useful incarrying out the methods of the invention, e.g, for maturing humanhepatocyte-like cells in accordance with the invention. A kit accordingto the invention comprises at least one activator of a retinoic acidresponsive receptor and at least one selected from GSK3 inhibitor,activator of Wnt signalling, CDK inhibitor and extracellular matrix(ECM) component or ECM component mixture.

Accordingly, a kit of the invention may comprise at least one activatorof a retinoic acid responsive receptor, at least one GSK3 inhibitor, andoptionally at least one extracellular matrix (ECM) component or ECMcomponent mixture.

A kit of the invention may also comprise at least one activator of aretinoic acid responsive receptor, at least one activator of Wntsignalling, and optionally at least one extracellular matrix (ECM)component or ECM component mixture.

A kit of the invention may also comprise at least one activator of aretinoic acid responsive receptor, at least one CDK inhibitor, andoptionally at least one extracellular matrix (ECM) component or ECMcomponent mixture.

A kit of the invention may also comprise at least one activator of aretinoic acid responsive receptor, at least one CDK inhibitor and atleast one GSK3 inhibitor or activator of Wnt signalling, and optionallyat least one extracellular matrix (ECM) component or ECM componentmixture.

A kit of the invention may also comprise at least one activator of aretinoic acid responsive receptor and at least one extracellular matrix(ECM) component or ECM component mixture.

As noted above, it is understood that the details given herein withrespect to the components employed in the methods of the invention alsoapply to the components comprised by the kits of the invention.

Hence, the at least one activator of a retinoic acid responsive receptorcomprised by a kit of the invention may, for instance, be a retinoicacid, such as 9-cis-retinoic acid.

The at least one GSK-3 inhibitor comprised by a kit of the inventionmay, for instance, be one selected from Kenpaullone 1-Aza-Kenpaullone,Alsterpaullone, Aminopyrimidine CHIR99021 and Indirubin-3′-monoxime.

The at least one GSK-3 inhibitor comprised by a kit of the inventionmay, for instance, be one selected from Kenpaullone, 1-Aza-Kenpaullone,Alsterpaullone, Indirubin-3′-monoxime. 2-bromo-9-nitropaullone,2-bromo-9-trifluoromethylpaullone, 2-bromopaullone,2-iodo-9-trifluoromethylpaullone, 2-iodopaullone,2-phenyl-4-(2-thienyl)-5H-pyrido[2±3-d][1]benzazepine-6(7H)-thione,2-[2-(1-hydroxycyclohexyl)-ethinyl]-9-trifluoromethyl-paullone,2,3-dimethoxy-9-nitropaullone, 2,3-dimethoxy-9-trifluormethylpaullone,2,3-dimethoxypaullone,2-(3-hydroxy-1-propinyl)-9-trifluoromethylpaullone,2,9-dibromo-paullone,3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-propionitrile,4-methoxypaullone,4-(4-chlorophenyl)-2-(2-naphthyl)-5H-pyrido[2±3-d][1]benzazepine-6(7H)-thione,5-benzyl-9-bromopaullone, 5-iodo-indirubin.3-monoxime,5,6,7,12-tetrahydro-benzo[6±7]cyclohept[1,2-b]indole, 6 bromo indirubin,8,10-dichloropaullone, 9-bromo-2,3-dihydroxypaullone,9-bromo-2,3-dimethoxypaullone, 9-bromo-4-hydroxypaullone,9-bromo-4-methoxypaullone,9-bromo-5-ethylpaullone,9-bromo-5-(methyloxycarbonylmethyl)paullone,9-bromo-5-methylpaullone,9-bromo-5,6,7,12-tetrahydro-benzo[6±7]cyclohept[1,2-b]indole,9-bromo-5,7-bis-(tert.-butyloxycarbonyl)-paullone,9-bromo-5,7,12-tri-(tert.-butyloxycarbonyl)-paullone,9-bromo-5,12-bis-(tert.-butyloxycarbonyl)-paullone,9-bromo-7,12-dihydro-6-(hydroxyamino)-indolo[2±3-d][1]benzazepine,9-bromo-7,12-dihydro-6-methylthio-indolo[2±3-d][1]benzazepine,9-bromo-7,12-dihydro-indolo[2±3-d][1]benzazepine-6(5H)-thione,9-bromo-12-(2-hydroxyethyl)-paullone, 9-bromo-12-(2-propenyl)-paullone,9-bromo-12-ethylpaullone, 9-bromo-12-methylpaullone,9-bromo-12-methyloxycarbonyl-methylpaullone,9-bromo-12-(tert.-butyloxycarbonyl)-paullone, 9-chloropaullone,9-cyanopaullone, 9-cyano-2,3-dimethoxypaullone, 9-fluoropaullone,9-methoxypaullone, 9-methylpaullone,9-oxo-thiazolo[5,4-f]quinazoline-2-carbonitril derivatives,9-trifluoromethylpaullone, 10-bromopaullone, 11-bromopaullone,11-chloropaullone, 11-ethylpaullone, 11-methylpaullone, Aloisines(=6-phenyl[5H]pyrrolo[2,3-6]pyrazines); e.g. Aloisine A, AZD1080,bis-indole indirubin, Debromohymenialdisine, Dibromocantharelline,(E)-3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-acrylicacid methyl ester, (E)-2-(3-oxo-1-butenyl)-9-trifluoromethylpaullone,(E)-3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-acrylonitrile,Indirubin, Hymenidin, Hymenialdisine, Manzamines, e.g. Manzamine A,Meridiamins, Paullone, Pyrazolo[3,4-b]pyridine derivatives,Pryazolo[3,4-b]quinoxaline derivatives andthiazolo[5,4-f]quinazolino-9-one derivatives.

The at least one activator of Wnt signalling comprised by a kit of theinvention may, for instance, be one selected from the group of Wntproteins disclosed above. It may, for instance, be Wnt3A or Wnt5A.

The at least one CDK inhibitor comprised by a kit of the invention may,for instance, be one selected from Kenpaullone, 1-Aza-Kenpaullone,Alsterpaullone, Indirubin-3′-monoxime, SNS-032(BMS-387032), AT-7519 andAZD5438.

The at least one CDK inhibitor comprised by a kit of the invention may,for instance, be one selected from Kenpaullone, 1-Aza-Kenpaullone,Alsterpaullone, Indirubin-3′-monoxime. 2-bromo-9-nitropaullone,2-bromo-9-trifluoromethylpaullone, 2-bromopaullone,2-iodo-9-trifluoromethylpaullone, 2-iodopaullone,2-phenyl-4-(2-thienyl)-5H-pyrido[2±3-d][1]benzazepine-6(7H)-thione,2-[2-(1-hydroxycyclohexyl)-ethinyl]-9-trifluoromethyl-paullone,2,3-dimethoxy-9-nitropaullone, 2,3-dimethoxy-9-trifluormethylpaullone,2,3-dimethoxypaullone,2-(3-hydroxy-1-propinyl)-9-trifluoromethylpaullone,2,9-dibromo-paullone,3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-propionitrile,4-methoxypaullone,4-(4-chlorophenyl)-2-(2-naphthyl)-5H-pyrido[2±3-d][1]benzazepine-6(7H)-thione,5-benzyl-9-bromopaullone, 5-iodo-indirubin.3-monoxime,5,6,7,12-tetrahydro-benzo[6±7]cyclohept[1,2-b]indole, 6 bromo indirubin,8,10-dichloropaullone, 9-bromo-2,3-dihydroxypaullone,9-bromo-2,3-dimethoxypaullone, 9-bromo-4-hydroxypaullone,9-bromo-4-methoxypaullone,9-bromo-5-ethylpaullone,9-bromo-5-(methyloxycarbonylmethyl)paullone,9-bromo-5-methylpaullone,9-bromo-5,6,7,12-tetrahydro-benzo[6±7]cyclohept[1,2-b]indole,9-bromo-5,7-bis-(tert.-butyloxycarbonyl)-paullone,9-bromo-5,7,12-tri-(tert.-butyloxycarbonyl)-paullone,9-bromo-5,12-bis-(tert.-butyloxycarbonyl)-paullone,9-bromo-7,12-dihydro-6-(hydroxyamino)-indolo[2±3-d][1]benzazepine,9-bromo-7,12-dihydro-6-methylthio-indolo[2±3-d][1]benzazepine,9-bromo-7,12-dihydro-indolo[2±3-d][1]benzazepine-6(5H)-thione,9-bromo-12-(2-hydroxyethyl)-paullone, 9-bromo-12-(2-propenyl)-paullone,9-bromo-12-ethylpaullone, 9-bromo-12-methylpaullone,9-bromo-12-methyloxycarbonyl-methylpaullone,9-bromo-12-(tert.-butyloxycarbonyl)-paullone, 9-chloropaullone,9-cyanopaullone, 9-cyano-2,3-dimethoxypaullone, 9-fluoropaullone,9-methoxypaullone, 9-methylpaullone,9-oxo-thiazolo[5,4-f]quinazoline-2-carbonitril derivatives,9-trifluoromethylpaullone, 10-bromopaullone, 11-bromopaullone,11-chloropaullone, 11-ethylpaullone, 11-methylpaullone, Aloisines(=6-phenyl[5H]pyrrolo[2,3-6]pyrazines); e.g. Aloisine A, AZD1080,bis-indole indirubin, Debromohymenialdisine, Dibromocantharelline,(E)-3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-acrylicacid methyl ester, (E)-2-(3-oxo-1-butenyl)-9-trifluoromethylpaullone,(E)-3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-acrylonitrile,Indirubin, Hymenidin, Hymenialdisine, Manzamines, e.g. Manzamine A,Meridiamins, Paullone, Pyrazolo[3,4-b]pyridine derivatives,Pryazolo[3,4-b]quinoxaline derivatives andthiazolo[5,4-f]quinazolino-9-one derivatives.

The at least one ECM component comprised by a kit of the invention may,for instance, be one selected from collagen, such as collagen I, II,III, IV, V or VI, fibronectin, elastin, chondroitin sulfateproteoglycan, dermatan sulfate proteoglycan, heparin proteoglycan,heparan sulfate proteoglycan, such as glypicans, syndecans or perlecans,glycosaminoglycans, nidogen/entactin, laminins, biglycan, tenascin, andhyaluronans.

A kit of the invention may comprise a ECM component mixture comprising,or composed of, two, three, four, five, six, seven, eight, nine or ten,or up to 20 of the ECM components mentioned herein. Thus, a kit of theinvention may comprise a ECM component mixture comprising, or composedof, two of the ECM components mentioned herein. A kit of the inventionmay also comprise a ECM component mixture comprising, or composed of,three of the ECM components mentioned herein. A kit of the invention mayalso comprise a ECM component mixture comprising, or composed of, fourof the ECM components mentioned herein. A kit of the invention may alsocomprise a ECM component mixture comprising, or composed of, five of theECM components mentioned herein. A kit of the invention may alsocomprise a ECM component mixture comprising, or composed of, six of theECM components mentioned herein. A kit of the invention may alsocomprise a ECM component mixture comprising, or composed of, seven ofthe ECM components mentioned herein. A kit of the invention may alsocomprise a ECM component mixture comprising, or composed of, eight ofthe ECM components mentioned herein. A kit of the invention may alsocomprise a ECM component mixture comprising, or composed of, nine of theECM components mentioned herein. A kit of the invention may alsocomprise a ECM component mixture comprising, or composed of, ten of theECM components mentioned herein.

For example, a kit of the invention may comprise a ECM component mixturecomprising, or composed of, collagen and fibronectin, such as a ECMcomponent mixture comprising, or composed of, collagen I andfibronectin. A kit of the invention may comprise a ECM component mixturecomprising, or composed of, collagen, such as collagen I, and laminin. Akit of the invention may comprise a ECM component mixture comprising, orcomposed of, collagen, such as collagen I, nidogen and laminin. A kit ofthe invention may comprise a ECM component mixture comprising, orcomposed of, collagen, such as collagen I, fibronectin and laminin. Akit of the invention may comprise a ECM component mixture comprising, orcomposed of, fibronectin and laminin. A kit of the invention maycomprise a ECM component mixture comprising, or composed of,Fibronectin, nidogen and laminin.

A kit of the invention may comprise a ECM component mixture comprising,or composed of, fibronectin, collagen I, collagen IV, collagen VI,nidogen, biglycan and laminin.

A kit of the invention may comprise a ECM component mixture comprising,or composed of, fibronectin, collagen I, collagen IV, nidogen, biglycanand laminin.

A kit of the invention may comprise a ECM component mixture comprising,or composed of, collagen, such as collagen I, laminin, fibronectin,proteoglycans and glycosaminoglycans. A kit of the invention maycomprise a ECM component mixture comprising, or composed of, collagen,such as collagen I, laminin, fibronectin, and proteoglycans.

A kit of the invention may comprise a ECM component mixture comprising,or composed of, collagen, such as collagen I, laminin, fibronectin,tenascin, elastin, proteoglycans and glycosaminoglycans.

Accordingly, a kit of the invention may comprise 9-cis-retinoic acid andKenpaullone. Such kit may further comprise a ECM component mixture asdisclosed above, such as, a ECM component mixture comprising, orcomposed of, collagen and fibronectin, such as a ECM component mixturecomprising, or composed of, collagen I and fibronectin.

A kit of the invention may comprise 9-cis-retinoic acid andIndirubin-3′-monoxime. Such kit may further comprise a ECM componentmixture as disclosed above, such as, a ECM component mixture comprising,or composed of, collagen and fibronectin, such as a ECM componentmixture comprising, or composed of, collagen I and fibronectin.

A kit of the invention may further comprise at least one DNAdemethylating agent. The DNA demethylating agent, if comprised by thekit of the invention, may be a cytidine analogue, such as e.g.5-aza-2-deoxycytidine (decitabine), 5-azacytidine (azacitidine),zebularine, Pseudoisocytidine, 5-fluoro-2-deoxycytidine,5,6-dihydro-5-azacytidine, 2′-deoxy-5,6-dihydro-5-azacytidine,6-azacytidine, 2′,2′-Difluoro-deoxycytidine (gemcitabine), orCytosine-beta-D-arabinofurasonide.

The at least one DNA demethylating agent may, for instance, be5-aza-2-deoxycytidine (decitabine). The at least one DNA demethylatingagent may also be 5-azacytidine (azacitidine).

A kit of the invention may further comprise human pluripotent stemcells. Hence, a kit of the invention may comprise human embryonic stemcells or human induced pluripotent stem cells. The human pluripotentstem cells may suitably be provided as a cell suspension, and may beprovided in a frozen state.

A kit of the invention may further comprise definitive endoderm cells(DE cells). The DE cells may suitably be provided as a cell suspension,and may be provided in a frozen state.

The components of a kit of the invention may be provided in the same orseparate containers. For instance, the at least one activator of aretinoic acid responsive receptor and the at least one GSK3 inhibitor,the at least one activator of Wnt signalling or the at least one CDKinhibitor may be provided in the same container. If an at least oneextracellular matrix (ECM) component or ECM component mixture is alsocomprised by the kit, such ECM component or ECM component mixture may begenerally provided in a separate container. Hence, while the at leastone activator of a retinoic acid responsive receptor and the at leastone GSK3 inhibitor, the at least one activator of Wnt signalling or theat least one CDK inhibitor may be provided in the same container, atleast one extracellular matrix (ECM) component or ECM component mixtureis provided in a different container.

Likewise, if human pluripotent stem cells or definitive endoderm cells(DE cells) are comprised by a kit, the human pluripotent stem cells orDE cells are generally provide in a container which is different fromthe container(s) containing the other components.

The invention also provides compositions. Such compositions areparticularly useful for maturing human hepatocyte-like cells inaccordance with the invention. A composition of the invention comprisesat least one activator of a retinoic acid responsive receptor and atleast one selected from GSK3 inhibitor, activator of Wnt signalling andCDK inhibitor.

Accordingly, a composition of the invention may comprise at least oneactivator of a retinoic acid responsive receptor and at least one GSK-3inhibitor, and optionally at least one CDK inhibitor.

A composition of the invention may also comprise at least one activatorof a retinoic acid responsive receptor and at least one activator of Wntsignalling, and optionally at least one CDK inhibitor.

A composition of the invention may also comprise at least one activatorof a retinoic acid responsive receptor and at least one CDK inhibitor,and optionally at least one GSK3 inhibitor or at least one activator ofWnt signalling.

As noted above, it is understood that the details given herein withrespect to the components employed in the methods of the invention alsoapply to the components comprised by the composition of the invention.

Hence, the at least one activator of a retinoic acid responsive receptorcomprised by a composition of the invention may, for instance, be aretinoic acid, such as 9-cis-retinoic acid.

The at least one GSK-3 inhibitor comprised by a composition of theinvention may, for instance, be one selected from Kenpaullone,1-Aza-Kenpaullone, Alsterpaullone, Aminopyrimidine CHIR99021 andIndirubin-3′-monoxime.

The at least one GSK-3 inhibitor comprised by a composition of theinvention may, for instance, be one selected from Kenpaullone,1-Aza-Kenpaullone, Alsterpaullone, Indirubin-3′-monoxime.2-bromo-9-nitropaullone, 2-bromo-9-trifluoromethylpaullone,2-bromopaullone, 2-iodo-9-trifluoromethylpaullone, 2-iodopaullone,2-phenyl-4-(2-thienyl)-5H-pyrido[2±3-d][1]benzazepine-6(7H)-thione,2-[2-(1-hydroxycyclohexyl)-ethinyl]-9-trifluoromethyl-paullone,2,3-dimethoxy-9-nitropaullone, 2,3-dimethoxy-9-trifluormethylpaullone,2,3-dimethoxypaullone,2-(3-hydroxy-1-propinyl)-9-trifluoromethylpaullone,2,9-dibromo-paullone,3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-propionitrile,4-methoxypaullone,4-(4-chlorophenyl)-2-(2-naphthyl)-5H-pyrido[2±3-d][1]benzazepine-6(7H)-thione,5-benzyl-9-bromopaullone, 5-iodo-indirubin.3-monoxime,5,6,7,12-tetrahydro-benzo[6±7]cyclohept[1,2-b]indole, 6 bromo indirubin,8,10-dichloropaullone, 9-bromo-2,3-dihydroxypaullone,9-bromo-2,3-dimethoxypaullone, 9-bromo-4-hydroxypaullone,9-bromo-4-methoxypaullone,9-bromo-5-ethylpaullone,9-bromo-5-(methyloxycarbonylmethyl)paullone,9-bromo-5-methylpaullone,9-bromo-5,6,7,12-tetrahydro-benzo[6±7]cyclohept[1,2-b]indole,9-bromo-5,7-bis-(tert.-butyloxycarbonyl)-paullone,9-bromo-5,7,12-tri-(tert.-butyloxycarbonyl)-paullone,9-bromo-5,12-bis-(tert.-butyloxycarbonyl)-paullone,9-bromo-7,12-dihydro-6-(hydroxyamino)-indolo[2±3-d][1]benzazepine,9-bromo-7,12-dihydro-6-methylthio-indolo[2±3-d][1]benzazepine,9-bromo-7,12-dihydro-indolo[2±3-d][1]benzazepine-6(5H)-thione,9-bromo-12-(2-hydroxyethyl)-paullone, 9-bromo-12-(2-propenyl)-paullone,9-bromo-12-ethylpaullone, 9-bromo-12-methylpaullone,9-bromo-12-methyloxycarbonyl-methylpaullone,9-bromo-12-(tert.-butyloxycarbonyl)-paullone, 9-chloropaullone,9-cyanopaullone, 9-cyano-2,3-dimethoxypaullone, 9-fluoropaullone,9-methoxypaullone, 9-methylpaullone,9-oxo-thiazolo[5,4-f]quinazoline-2-carbonitril derivatives,9-trifluoromethylpaullone, 10-bromopaullone, 11-bromopaullone,11-chloropaullone, 11-ethylpaullone, 11-methylpaullone, Aloisines(=6-phenyl[5H]pyrrolo[2,3-6]pyrazines); e.g. Aloisine A, AZD1080,bis-indole indirubin, Debromohymenialdisine, Dibromocantharelline,(E)-3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-acrylicacid methyl ester, (E)-2-(3-oxo-1-butenyl)-9-trifluoromethylpaullone,(E)-3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-acrylonitrile,Indirubin, Hymenidin, Hymenialdisine, Manzamines, e.g. Manzamine A,Meridiamins, Paullone, Pyrazolo[3,4-b]pyridine derivatives,Pryazolo[3,4-b]quinoxaline derivatives andthiazolo[5,4-f]quinazolino-9-one derivatives.

The at least one activator of Wnt signalling comprised by a compositionof the invention may, for instance, be one selected from the group ofWnt proteins disclosed above. It may, for instance, be Wnt3A or Wnt5A.

The at least one CDK inhibitor comprised by a composition of theinvention may, for instance, be one selected from Kenpaullone,1-Aza-Kenpaullone, Alsterpaullone, Indirubin-3′-monoxime,SNS-032(BMS-387032), AT-7519 and AZD5438.

The at least one CDK inhibitor comprised by a composition of theinvention may, for instance, be one selected from Kenpaullone,1-Aza-Kenpaullone, Alsterpaullone, Indirubin-3′-monoxime.2-bromo-9-nitropaullone, 2-bromo-9-trifluoromethylpaullone,2-bromopaullone, 2-iodo-9-trifluoromethylpaullone, 2-iodopaullone,2-phenyl-4-(2-thienyl)-5H-pyrido[2±3-d][1]benzazepine-6(7H)-thione,2-[2-(1-hydroxycyclohexyl)-ethinyl]-9-trifluoromethyl-paullone,2,3-dimethoxy-9-nitropaullone, 2,3-dimethoxy-9-trifluormethylpaullone,2,3-dimethoxypaullone,2-(3-hydroxy-1-propinyl)-9-trifluoromethylpaullone,2,9-dibromo-paullone,3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-propionitrile,4-methoxypaullone,4-(4-chlorophenyl)-2-(2-naphthyl)-5H-pyrido[2±3-d][1]benzazepine-6(7H)-thione,5-benzyl-9-bromopaullone, 5-iodo-indirubin.3-monoxime,5,6,7,12-tetrahydro-benzo[6±7]cyclohept[1,2-b]indole, 6 bromo indirubin,8,10-dichloropaullone, 9-bromo-2,3-dihydroxypaullone,9-bromo-2,3-dimethoxypaullone, 9-bromo-4-hydroxypaullone,9-bromo-4-methoxypaullone,9-bromo-5-ethylpaullone,9-bromo-5-(methyloxycarbonylmethyl)paullone,9-bromo-5-methylpaullone,9-bromo-5,6,7,12-tetrahydro-benzo[6±7]cyclohept[1,2-b]indole,9-bromo-5,7-bis-(tert.-butyloxycarbonyl)-paullone,9-bromo-5,7,12-tri-(tert.-butyloxycarbonyl)-paullone,9-bromo-5,12-bis-(tert.-butyloxycarbonyl)-paullone,9-bromo-7,12-dihydro-6-(hydroxyamino)-indolo[2±3-d][1]benzazepine,9-bromo-7,12-dihydro-6-methylthio-indolo[2±3-d][1]benzazepine,9-bromo-7,12-dihydro-indolo[2±3-d][1]benzazepine-6(5H)-thione,9-bromo-12-(2-hydroxyethyl)-paullone, 9-bromo-12-(2-propenyl)-paullone,9-bromo-12-ethylpaullone, 9-bromo-12-methylpaullone,9-bromo-12-methyloxycarbonyl-methylpaullone,9-bromo-12-(tert.-butyloxycarbonyl)-paullone, 9-chloropaullone,9-cyanopaullone, 9-cyano-2,3-dimethoxypaullone, 9-fluoropaullone,9-methoxypaullone, 9-methylpaullone,9-oxo-thiazolo[5,4-f]quinazoline-2-carbonitril derivatives,9-trifluoromethylpaullone, 10-bromopaullone, 11-bromopaullone,11-chloropaullone, 11-ethylpaullone, 11-methylpaullone, Aloisines(=6-phenyl[5H]pyrrolo[2,3-6]pyrazines); e.g. Aloisine A, AZD1080,bis-indole indirubin, Debromohymenialdisine, Dibromocantharelline,(E)-3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-acrylicacid methyl ester, (E)-2-(3-oxo-1-butenyl)-9-trifluoromethylpaullone,(E)-3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[2±3-d][1]benzazepin-2-yl)-acrylonitrile,Indirubin, Hymenidin, Hymenialdisine, Manzamines, e.g. Manzamine A,Meridiamins, Paullone, Pyrazolo[3,4-b]pyridine derivatives,Pryazolo[3,4-b]quinoxaline derivatives andthiazolo[5,4-f]quinazolino-9-one derivatives.

Accordingly, a composition of the invention may comprise 9-cis-retinoicacid and Kenpaullone.

A composition of the invention may comprise 9-cis-retinoic acid andIndirubin-3′-monoxime.

A composition of the invention may comprise 13-cis-retinoic acid andkenpaullone.

A composition of the invention may comprise 13-cis-retinoic acid andIndirubin-3′-monoxime.

Definitions

As used herein, “pluripotent” or “pluripotency” refers to the potentialto form all types of specialized cells of the three germ layers(endoderm, mesoderm, and ectoderm); and is to be distinguished from“totipotent” or “totipotency”, that is the ability to form a completeembryo capable of giving rise to offsprings.

As used herein, “human pluripotent stem cells” (hPSC) refers to humancells that have the capacity, under appropriate conditions, toself-renew as well as the ability to form any type of specialized cellsof the three germ layers (endoderm, mesoderm, and ectoderm). hPS cellsmay have the ability to form a teratoma in 8-12 week old SCID miceand/or the ability to form identifiable cells of all three germ layersin tissue culture. Included in the definition of human pluripotent stemcells are embryonic cells of various types including human embryonicstem (hES) cells, (see, e.g., Thomson et al. (1998), Heins et. al.(2004), as well as induced pluripotent stem cells [see, e.g. Takahashiet al., (2007); Zhou et al. (2009); Yu and Thomson in Essentials of StemCell Biology (2^(nd) Edition]. The various methods described herein mayutilise hPS cells from a variety of sources. For example, hPS cellssuitable for use may have been obtained from developing embryos by useof a non-destructive technique such as by employing the singleblastomere removal technique described in e.g. Chung et al (2008),further described by Mercader et al. in Essential Stem Cell Methods(First Edition, 2009). Additionally or alternatively, suitable hPS cellsmay be obtained from established cell lines or may be adult stem cells.

As used herein “hiPS cells” refers to human induced pluripotent stemcells. hiPS cells are a type of pluripotent stem cells derived fromnon-pluripotent cells—typically adult somatic cells—by induction of theexpression of genes associated with pluripotency, such as SSEA-3,SSEA-4, TRA-1-60, TRA-1-81, Oct-4, Sox2, Nanog and Lin28.

As used herein “definitive endoderm (DE)” and “definitive endoderm cells(DE cells)” refers to cells exhibiting protein and/or gene expression aswell as morphology typical to cells of the definitive endoderm or acomposition comprising a significant number of cells resembling thecells of the definitive endoderm. The definitive endoderm is the germcell layer which gives rise to cells of the intestine, pancreas, liverand lung. DE cells may generally be characterized, and thus identified,by a positive gene and protein expression of the endodermal markersFOXA2, CXCR4, HHEX, SOX17, GATA4 and GATA6. The two markers SOX17 andCXCR4 are specific for DE and not detected in hPSC, hepatic progenitorcells or hepatocytes. Lastly, DE cells do not exhibit gene and proteinexpression of the undifferentiated cell markers Oct4, SSEA-3, SSEA-4,TRA-1-60, TRA-1-81, but can show low Nanog expression.

As used herein, “hepatic progenitors” or “hepatic progenitor cells”refers to cells which have entered the hepatic cell path and give riseto hepatocyte. “Hepatic progenitors” are thus distinguished from“endodermal cells” in that they have lost the potential to develop intocells of the intestine, pancreas and lung. “Hepatic progenitors” maygenerally be characterized, and thus identified, by a positive gene andprotein expression of the early hepatic markers EpCAM, c-Met(HGF-receptor), AFP, CK19, HNF6, C/EBPα and β. They do not exhibit geneand protein expression of the DE-markers CXCR4 and SOX17. Lastly,“hepatic progenitors” do not exhibit gene and protein expression of theundifferentiated cell markers Oct4, SSEA-3, SSEA-4, TRA-1-60 andTRA-1-81 nor the mature hepatic markers CYP1A2, CYP2C9, CYP19, CYP3A4,CYP2B6 and PXR.

As used herein, “hepatocyte” or “hepatocyte-like cells” refers to fullydifferentiated hepatic cells. “Hepatocytes” or “hepatocytes-like cells”may generally be described, and thus identified, by a positive gene andprotein expression of the mature hepatic markers CYP1A2, CYP3A4, CYP2C9,CYP2C19, CYP2B6, GSTA1-1, OATP-2, NTCP, Albumin, PXR, CAR, and HNF4a(isoforms 1+2) among others. Further, “hepatocytes” or “hepatocyte-likecells do not exhibit gene and protein expression of the undifferentiatedcell markers Oct4, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Compared to DEcells, “hepatocytes” or “hepatocyte-like cells do not exhibit gene andprotein expression of the DE cell markers SOX17 and CXCR4. Compared to“hepatic progenitors”, “hepatocytes” or “hepatocyte-like cells do notexhibit gene and protein expression of the hepatic progenitor markersCytokeratin 19 and AFP.

As meant herein, a gene or protein shall be interpreted as being“expressed”, if in an experiment measuring the expression level of saidgene or protein, the determined expression level is higher than threetimes the standard deviation of the determination, wherein theexpression level and the standard deviation are determined in 10separate determinations of the expression level. The determination ofthe expression level in the 10 separate determinations is preferablycorrected for background-signal.

As used herein HDAC inhibitors refers to Histone deacetylase inhibitors,such as Sodium Butyrate (“NaB”), Phenyl Butyrate (“PB”), Trichostatin Aand Valproic Acid (“VA”).

As used herein, “GSK inhibitor” refers to a compound which inhibits GSK(especially GSK3, including GSK3alpha or GSK3beta).

As used herein, “activator of Wnt signalling” refers to a compound whichactivates Wnt signalling.

As used herein, a DNA demethylating agent is intended to mean a compoundthat interferes with DNA methyltransferase enzyme activity, such asnucleoside analogues, like cytidine analogs, notably5-aza-2-deoxycytidine (decitabine) and 5-azacytidine (azacitidine), andnon-nucleoside types, such as RG108, S-5-Adenosyl-L-homocysteine, andprocaine.

As used herein “CYP” is intended to mean Cytochrome P, and morespecifically Cytochrome P 450, the major phase I metabolizing enzyme ofthe liver constituting of many different isoenzymes, such as CYP1A1,CYP1A2, CYP1B1, CYP2A6/2A7/2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C19,CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7 and CYP7A1.

As used herein, the term “GST” is intended to mean glutathionetransferase, and examples of subtypes thereof are GST A1-1, GST M1-1,and GST P1-1.

As used herein the term “UGT” is intended to mean uridinediphosphoglucuronosyltransferase, which is a group of liver enzymescatalysing glucuronidation activities.

As used herein the term “NTCP” is taken to mean Na+-taurocholatecotransporting polypeptide, a Sodium/bile acid co-transporter encoded bythe gene SLC10A1

As used herein the term “CAR” is taken to mean Constitutive androstanereceptor

The term “functional drug metabolising enzymes” is intended to meanfunctional enzymes belonging to the phase I and phase II enzymes thatperform chemical modifications of xenobiotics and drugs, so called drugor xenobiotic metabolism.

As used herein, the term “functional activity” means effectivemeasurable hepatic cell function, such as a measurable transportation ofdrugs for drug transporters and a measurable metabolism of enzymes forthe Cytochrome P450s (CYPs), commonly detected in primary humanhepatocytes.

As used herein, the term “extraembryonic endoderm (ExE)” is intended tomean the differentiated endodermal cells that, as to the opposite of thedefinitive endoderm, will constitute the compartments outside the embryoin the human development, such as the yolk sac.

As used herein, the term “AAT” is intended to mean the liver markeralpha-anti-trypsin.

As used herein, the term “AFP” is intended to mean the liver markeralpha-fetoprotein.

As used herein, the term “BSEP” is intended to mean the bile transporterbile salt export pump.

As used herein, the term “CK” is intended to mean the liver markercytokeratin (used interchangeably) with different subtypes such asCytokeratin 18 (CK18/KRT18), Cytokeratin 19 (CK19/KRT19), Cytokeratin 8(CK8) and Cytokeratin 7 (CK7).

As used herein, the term “FGF” means fibroblast growth factor,preferably of human and/or recombinant origin, and subtypes belongingthereto are e.g. “bFGF” (means basic fibroblast growth factor, sometimesalso referred to as FGF2) and FGF4. “aFGF” means acidic fibroblastgrowth factor (sometimes also referred to as FGF1).

As used herein, the term “BMP” means Bone Morphogenic Protein,preferably of human and/or recombinant origin, and subtypes belongingthereto are e.g. BMP4 and BMP2.

As used herein, the term “HGF” means Hepatocyte Growth Factor,preferably of human and/or recombinant origin.

As used herein, the term “EGF” means Epidermal Growth Factor, preferablyor human and/or recombinant origin.

As used herein, the “HNF4alpha”, or “HNF4a”, used interchangeably areintended to mean hepatocyte nuclear factor 4 also known as NR2A1(nuclear receptor subfamily 2, group A, member 1), a transcriptionfactor regulating gene expression in endodermal derived tissue, e.g. theliver, pancreatic islets, and adipocytes. The encoded protein controlsthe expression of several genes, including hepatocyte nuclear factor 1alpha.

As used herein, the term “MDR” is intended to mean multi-drug resistancetransporter. MDR 1 and 3 are members of the ATP-binding cassette (ABC)family of transporters and both are drug efflux transporters. MDR 1 isimportant in regulating the traffic of drugs, peptides and xenobioticsinto the body and in protecting the body against xenobiotic insults anddrug toxicity, while MDR 3 is essential for phospholipid secretion intobile.

As used herein, the term “Activin” is intended to mean a TGF-beta familymember that exhibits a wide range of biological activities includingregulation of cellular proliferation and differentiation such as“Activin A” or “Activin B”. Activin belongs to the common TGF-betasuperfamiliy of ligands.

As used herein, the term “activator of a retinoic acid responsivereceptor” is intended to mean a compound capable of binding to andactivating a human retinoic acid receptor (RAR) and/or retinoid Xreceptor (RXRs).

As used herein, the term “retinoic acid receptor” or “RAR” is intendedto mean a member of the family of retinoic acid receptors, in particularRAR-alpha, RAR-beta, and RAR-gamma, which are encoded by the RARA, RARB,RARG genes, respectively. Each receptor isoform has several splicevariants: two for alpha, four for beta, and two for gamma. Theseisoforms are also included in the definition of a “retinoic acidreceptor”.

As used herein, the term “retinoid X receptor” is intended to mean amember of the family of retinoid X receptors, in particular RXR-alpha,RXR-beta, and RXR-gamma, which are encoded by the RXRA, RXRB, RXRGgenes, respectively.

As used herein, the term “retinoic acid” is intended to mean a retinoicacid isomer, including but not limited to all-trans-retinoic acid,7-cis-retinoic acid, 9-cis retinoic acid, 11-cis-retinoic acid and13-cis retinoic.

As used herein, the term “inhibitor of a cyclin dependent kinase” or“CDK inhibitor” is intended to mean a compound capable of inhibiting thefunction (e.g., the activity) of a cyclin dependent kinase, such ascyclin dependent kinase 2 (CDK2).

As used herein, the term “ROCK inhibitor” is intended to mean aninhibitor of ROCK Rho-associated protein kinase activity

As used herein, the term “matrix” is intended to refer to any component,either isolated or in combination, which forms part of the normalmammalian extracellular matrix environment. Such matrix componentsinclude, but are not limited to, collagen, fibronectin, and laminin andmay be from natural or synthetic sources.

As used herein, the term “overlay” is intended to refer to a layer of,e.g., extracellular matrix components, which is applied on top of thecultured cells.

As used herein, the term “coating” is intended to refer to a layer of,e.g., extracellular matrix components, which covers the surface of aculture vessel and on which the cells are cultured.

As used herein the term “xeno-free” is intended to mean completecircumvention of direct or in-direct exposure to non-human animalcomponents.

As used herein, the term “hepatocellular toxicity” indicates cellularresponses such as necrotic toxicity, apoptosis, mitochondrial toxicity,phospholipidosis, steatosis and bile acid transport.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a, 1b and 1c show the expressions of HNF4α in hESC-derivedhepatocytes exposed to varying lengths of retinoic acid treatments.

FIGS. 2a, 2b, 2c and 2d show the functional expressions of several CYPenzymes in hESC-derived hepatocytes exposed to varying lengths ofretinoic acid treatments.

FIGS. 3a, 3b, 3c, 3d and 3e show the gene expressions of CYP enzymes,the nuclear receptors CAR and PXR as well as α-fetoprotein inhESC-derived hepatocytes exposed to varying lengths of retinoic acidtreatments.

FIGS. 4a, 4b and 4c show the functional expressions of CYP enzymes inhESC and hiPS-derived hepatocytes exposed to retinoic acid incombination with Kenpaullone and matrix overlay.

FIGS. 5a, 5b, 5c, 5d, 5e and 5f show the gene expressions of phase Ienzymes, phase II enzymes, drug transporters and nuclear receptors inhESC-derived hepatocyte-like cells (derived with basic protocol B)exposed to retinoic acid, Kenpaullone and a matrix overlay.

FIGS. 6a, 6b, 6c, 6d, 6e, 6f and 6g show the gene expressions of phase Ienzymes, phase II enzymes, drug transporters and nuclear receptors inhESC- and hiPSC-derived hepatocyte-like cells (derived with basicprotocols C and D) exposed to retinoic acid, Kenpaullone and a matrixoverlay.

FIGS. 7a, 7b and 7c show the morphology and longevity of hESC-derivedhepatocytes (derived with basic protocol B) treated with a matrixoverlay (protocol B).

FIGS. 8a, 8b, 8c and 8d show the morphology and longevity of hESC- andhiPSC-derived hepatocytes (derived with basic protocol C and D) exposedto retinoic acid, Kenpaullone and a matrix overlay.

FIGS. 9a, 9b, 9c, 9d, 9e, 9f and 9g are gene expressions of phase Ienzymes, phase II enzymes, drug transporters and nuclear receptors inhESC- and hiPSC-derived hepatocyte-like cells (derived with basicprotocols C and D) treated early with 5-aza-deoxycytidine and exposedlate to retinoic acid, Kenpaullone and a matrix overlay.

FIGS. 10a and 10b are functional expressions of CYP enzymes in hESC- andhiPSC-derived hepatocyte-like cells (derived with basic protocols C andD) treated early with 5-aza-deoxycytidine and exposed late to retinoicacid, Kenpaullone and a matrix overlay.

FIGS. 11a, 11b, 11c, 11d, 11e and 11f show morphology and longevity ofhESC- and hiPSC-derived hepatocyte-like cells (derived with basicprotocols C and D) treated early with 5-aza-deoxycytidine and exposedlate to retinoic acid, Kenpaullone and a matrix overlay.

FIGS. 12a and 12b show comparisons of functional expressions of CYPenzymes in hESC- and hiPSC-derived hepatocyte-like cells (derived withbasic protocols C and D) with and without early treatment with5-aza-deoxycytidine and with and without late exposure to retinoic acid,Kenpaullone and a matrix overlay.

FIGS. 13a 1, 13 a 2, 13 b 1, 13 b 2, 13 c 1, 13 c 2, 13 d 1, 13 d 2, 13d 3, 13 d 4, 13 d 5, 13 d 6 and 13 d 7 show the characterizations ofhESC- and hiPSC-derived definitive endodermal cells (derived with basicprotocols C and D) with and without a 5-aza-deoxycytidine treatmentduring the pre-endodermal phase (day 0-7 of the protocol).

FIGS. 14a, 14b, 14c and 14d show the characterizations of definitiveendodermal cells derived from 27 different hESC- and hiPSC lines(derived with basic protocols C and D, respectively) with a5-aza-deoxycytidine treatment during the pre-endodermal phase (day 0-7of the protocol).

FIGS. 15a, 15b, 15c and 15d show the characterizations of definitiveendodermal cells derived from 3 hESC- and hiPSC lines (derived withbasic protocols C and D, respectively) with or without a treatment with5-aza-deoxycytidine or 5-azacytidine during the pre-endodermal phase(day 0-7 of the protocol).

FIGS. 16a 1, 16 a 2, 16 a 3, 16 a 4, 16 a 5, 16 a 6, 16 b 1, 16 b 2, 16b 3 and 16 b 4 show comparisons of CYP enzyme expression incryopreserved human primary hepatocytes and in hESC- and hiPSC-derivedhepatocyte-like cells (derived with basic protocols C and D) with earlytreatment with 5-aza-deoxycytidine and late exposure to retinoic acid,Kenpaullone and a matrix overlay.

FIGS. 17a, 17b, 17c, 17d and 17e show the functional expressions of CYPenzymes in hiPSC-derived hepatocyte-like cells (derived with basicprotocols C and D) with early treatment with 5-aza-deoxycytidine andlate exposure to one or two activators of a retinoic acid responsivereceptor, Kenpaullone and a matrix overlay.

FIGS. 18a, 18b, 18c, 18d and 18e show the gene expressions of CYPenzymes and the nuclear receptor PXR in hiPSC-derived hepatocyte-likecells (derived with basic protocols C and D) with early treatment with5-aza-deoxycytidine and late exposure to one or two activators of aretinoic acid responsive receptor, Kenpaullone and a matrix overlay.

FIGS. 19a, 19b and 19c show the functional expressions of CYP enzymes inhiPSC-derived hepatocyte-like cells (derived with basic protocols C andD) treated early with 5-aza-deoxycytidine and exposed late to retinoicacid, Kenpaullone and a simple or more complex matrix overlay.

FIG. 20 shows the functional expression of CYP2C9 enzyme inhiPSC-derived hepatocyte-like cells (derived with basic protocol D)treated early with 5-aza-deoxycytidine and exposed late to Kenpaullone,9cis retinoic acid, or an analogue to 9cis retinoic acid.

FIGS. 21a 1, 21 a 2, 21 b 1 and 21 b 2 show the functional expressionsof CYP2C9 and 3A enzyme in hiPSC and hESC-derived hepatocyte-like cells(derived with basic protocols C and D) treated early with5-aza-deoxycytidine and exposed late to 9cis retinoic acid, Kenpaulloneor an analogue to Kenpaullone.

EXAMPLES

Examples of general culturing and passaging techniques are disclosed inapplications WO2004/099394, WO2003/055992, WO/2007/042225, WO2007/140968and WO2011116930.

As laid out in the following examples, the starting material maycomprise any hepatic progenitor cell type, particularly one derivedthrough an initial differentiation towards a definitive orextraembryonic lineage from a human pluripotent stem cell. The startingmaterial may also be any cell of hepatic progenitor lineage.

Example 1: Maintenance of hPS Cell Types

All hPS cells (as defined above) can be used as staring material forthis invention. For the examples below in particular hepatocyte-likecells were derived in vitro from undifferentiated human embryonic stemcells (hESC) established on mEF feeder cells (Heins et al 2004) andmaintained under feeder-free conditions. The cell lines used for thisexperiment could be, but are not limited to the hES cell lines SA167,SA181, SA461 (Cellartis AB, Göteborg, Sweden) and they can be propagatedas described by Heins et al. 2004. These cell lines are listed in theNIH stem cell registry, the UK Stem Cell bank and the European hESCregistry and are available on request.

Along with hPS obtained from hESC, hiPS (human induced pluripotent stem)cells have also been used for the derivation of hepatocytes for theexamples of this invention.

The hiPSC line used in this invention was derived as followed: Humandermal fibroblasts (CRL2429, ATCC) were maintained in DMEM supplementedwith 10% fetal bovine serum, 1× glutamax, 5 U/ml penicillin and 5 μg/mlstreptomycin at 37° C. in a humidified atmosphere of 5% CO₂ in air.Fibroblasts were tranduced with recombinant lentiviruses encoding mouseOct4, Sox2, Klf4 and c-myc and cultured for 5 days. The transduced cellswere then dispersed with trypsin and seeded onto mitomycin C treatedhuman dermal fibroblast feeder cells at a density of 5×10³ cells/cm² intheir normal growth medium. After 24 hours the medium was replaced withknockout DMEM supplemented with 20% knockout serum replacement, 1×non-essential amino acids, 1× glutamax, 5 U/ml penicillin, 5 μg/mlstreptomycin, 100 μM 2-mercaptoethanol and 30 ng/ml bFGF at 37° C. in ahumidified atmosphere of 5% CO₂ in air. Half of the volume of medium wasreplaced every day and colonies of iPS cells emerged after approximately30 days. iPS colonies were picked, expanded in DEF-CS™, and cell banksprepared. The banked cells were then characterised to check for theexpression of endogenous Oct4, Sox2, Klf4 and c-Myc, silencing oftransgenes, potential to differentiate into cell types representative ofall three germ layers in vitro, and to confirm their authenticity by STRprofiling and comparison with the parental fibroblast cell line (ATCC).Alternatively to reprogramming using lentivirus, hiPSC lines can also bereprogrammed using retrovirus, Sendai virus, adenovirus, episomalplasmid vectors, proteins and mRNAs or other techniques. Other suitablecell lines for use are those established by Chung et al. (2008), such ascell lines MA126, MA127, MA128 and MA129 (Advanced Cell Technology, Inc.Worcester, Mass., USA), which all are listed with the International stemcell registry. These cell lines have been derived (or obtained) withoutdestruction of the human embryo by employing a single blastomere removaltechnique.

Example 2: Differentiation of hPS Cell Types to Produce Hepatocyte-Like

Hepatocyte-like cells may be derived from hPS cells by employing thefollowing exemplary basic protocols A, B, C, and D:

Protocol A:

Undifferentiated hPS cells are dissociated and seeded directly infreshly prepared day 0-medium. The different mediums were preparedfreshly and added day 0, 1, 2, 3, 4, 5, 7 and then every second or thirdday during the pre-hepatic phase, and differentiation and maturationphase.

Day 0 RPMI 1640 (+0.1% PEST, +1% Glutamax) 1×B27

100 ng/ml Activin A

1 mM NaB

5 μM ROCK inhibitor

Day 1 RPMI 1640 (+0.1% PEST, +1% Glutamax) 1×B27

100 ng/ml Activin A

1 mM NaB Day 2-7 RPMI 1640 (+0.1% PEST+1% Glutamax) 1×B27

100 ng/ml Activin A

0.5 mM NaB

On day 7 the cells are passaged. The cells are incubated for 3-7 minuteswith TrypLE Select at 37° C., the same volume of VitroHES is added andthe cell suspension is centrifuged at 200-300 g, 5-6 min. Thereafter,the cells are replated onto a Gelatine-based coating at a cell densityof 50 000-350 000 cells/cm² such as e.g. 100 000-300 000 cells/cm²,preferably 150 000 cells/cm².

Day 7-14 (Pre-Hepatic) VitroHES 1% DMSO Day 14-45 (Differentiation andMaturation) WME+SQ (−GA1000)+1% Glutamax+0.1% PEST 0.1 μM DexM

10 ng/ml OsM20 ng/ml HGF

0.5% DMSO

1.4 μM (2′Z,3′£)-6-Bromoindirubin-3′-oxime (BIO)

Protocol B:

Undifferentiated hPS cells are dissociated and seeded directly infreshly prepared day 0-medium. The different mediums were preparedfreshly and added day 0, 1, 2, 3, 4, 5, 7 and then every second or thirdday during the pre-hepatic phase, and differentiation and maturationphase.

Day 0 RPMI 1640 (+0.1% PEST, +1% Glutamax) 1×B27

100 ng/ml Activin A

1 mM NaB

5 μM ROCK inhibitor

Day 1 RPMI 1640 (+0.1% PEST, +1% Glutamax) 1×B27

100 ng/ml Activin A

1 mM NaB 3 μM CHIR99021 Day 2-7 RPMI 1640 (+0.1% PEST+1% Glutamax) 1×B27

100 ng/ml Activin A

0.5 mM NaB

On day 7 the cells are passaged. The cells are incubated for 3-7 minuteswith TrypLE Select at 37° C., the same volume of VitroHES is added andthe cell suspension is centrifuged at 200-300 g, 5-6 min. Thereafter,the cells are replated onto a Fibronectin-based coating at a celldensity of 50 000-350 000 cells/cm² such as e.g. 100 000-300 000cells/cm², preferably 150 000 cells/cm². For media d7-14 (pre-hepatic)and 14-45 (differentiation and maturation) see Protocol A.

Protocol C:

Undifferentiated hPS cells are dissociated and seeded directly infreshly prepared day 0-medium. The different mediums were preparedfreshly and added day 0, 1, 2, 3, 4, 5, 7 and then every second or thirdday during the pre-hepatic phase, and differentiation and maturationphase. The pre-treatment medium is available from Cellectis AB (ArvidWallgrens Backe 20, 41346 Gothenburg, Sweden).

Day 0

Pre-treatment medium

3 μM CHIR99021

5 μM ROCK inhibitor

Day 1

Pre-treatment medium

3 μM CHIR99021 Day 2 RPMI 1640 (+0.1% PEST+1% Glutamax) 1×B27

50 ng/ml Activin A

3 μM CHIR99021 5 μM LY294002 Day 3 RPMI 1640 (+0.1% PEST+1% Glutamax)1×B27

50 ng/ml Activin A

5 μM LY294002 Day 4-7 RPMI 1640 (+0.1% PEST+1% Glutamax) 1×B27

50 ng/ml Activin A

For passage d7, media d7-14 (pre-hepatic) and 14-45 (differentiation andmaturation) see Protocol B.

Protocol D:

Undifferentiated hPS cells are dissociated and seeded directly infreshly prepared day 0-medium. The different mediums were preparedfreshly and added day 0, 1, 2, 3, 4, 5, 7 and then every second or thirdday during the pre-hepatic phase, and differentiation and maturationphase. The pre-treatment medium is available from Cellectis AB (ArvidWallgrens Backe 20, 41346 Gothenburg, Sweden).

Day 0

Pre-treatment medium

3 μM CHIR99021

5 μM ROCK inhibitor

Day 1 RPMI 1640 (+0.1% PEST+1% Glutamax) 1×B27

50 ng/ml Activin A

3 μM CHIR99021 5 μM LY294002 Day 2 RPMI 1640 (+0.1% PEST+1% Glutamax)1×B27

50 ng/ml Activin A

5 μM LY294002 Day 3-7 RPMI 1640 (+0.1% PEST+1% Glutamax) 1×B27

50 ng/ml Activin A

For passage d7, media d7-14 (pre-hepatic) and 14-45 (differentiation andmaturation) see Protocol B.

Example 3: Effect of Treatment of hESC-Derived Hepatocyte-Like Cellswith 9 Cis-Retinoic Acid on Expression of Different HNF4α IsoformsProcedure:

Following the basic protocol A, hepatocyte-like cells derived from hEScells cultured on a Gelatin-based coating were treated with 1 or 2 μM 9cis-retinoic acid for 5 hr, 24 hr or 48 hr on day 23 of the protocol(i.e. on day 9 of the differentiation and maturation) or long term fromday 14 (i.e. starting day 1 of the differentiation and maturation phase)and onwards and analysed immediately after the exposure (FIG. 1 A, B, C)or 7 days later (Fig. C). Three different HNF4α-TaqMan assays were usedfor analysis of HNF4α expression: one assay detecting all 9 HNF4αisoforms, one assay detecting isoforms 1-3 (including the adult isoforms1 and 2) and one assay detecting isoforms 4-9 (including the fetalisoforms 7 and 8). Isoforms 3, 4, 5, 6 and 9 are not expressed at all invivo or at very low levels and can therefore be neglected.

Results:

A) 5 hr RA-exposure strongly increase expression of the adult HNF4a1-3isoforms, but also increase the expression of the fetal HNF4a4-9isoforms. 24-, 48 hr-exposure and continuous RA-treatment slightlyincrease the adult HNF4a1-3 isoforms and slightly decrease the fetalHNF4a4-9 isoforms making the ratio of 1-3 isoforms/4-9 isoforms moresimilar to hphep, see also FIG. 1 B.

B) Human primary hepatocytes (hp hep) have a high ratio of the adultHNF4a isoforms 1-3 to the fetal 4-9 isoforms, whereas HepG2 have a lowratio. 24 hr and 48 hr RA-exposure and long term/continuous treatmentincrease the 1-3/4-9 ratio of hESC-derived hepatocytes to levels similaras in hp hep. 5 hr exposures do not increase the 1-3/4-9 ratio sincealso expression of 4-9 isoforms increases (see FIG. 1A). hphep: averageof 7 batches freshly isolated hp hep. HepG2: average of 2 batches.

C) A 5 hr exposure with 1 μM RA increases the expression of HNF4αisoforms 1-3 immediately after the exposure (see also A), but 7 dayslater the expression of isoforms 1-3 is slightly lower in the RA-treatedcells than in the untreated control cells. The expression of isoforms4-9 is slightly lower in the RA-treated cells than in the controlimmediately after the exposure. 7 days later expression of fetal isoformis raised over control values.

Therefore the optimal culture conditions for producing an increase inthe adult isoforms of HNF4a with minimal increase or decrease in fetalisoforms on day 23 involve the continuous treatment or 24 hr or 48 hrexposures of 1 or 2 μM RA on d23, corresponding to an expression profileclosest to that of primary human hepatocytes (hp hep). The skilledperson wishing to produce cells with an unchanged expression profilemight instead select a 5 hr exposure to RA.

Example 4: Effect of Treatment of hESC-Derived Hepatic Progenitors andHepatocyte-Like Cells with 9 Cis-Retinoic Acid (RA) on CYP ActivityProcedure:

Following the basic protocol A, differentiating hES cell derived hepaticprogenitors and hepatocyte-like cells cultured on a Gelatin-basedcoating were treated with 1 μM 9-cis retinoic acid for 5, 24 or 48 hrexposures on days 21, 22 or 23 of the protocol (i.e. on day 7, 8 or 9 ofthe differentiation and maturation phase; FIG. 2 A, B, D), repeated 5 hrexposures on days 11, 16, 23, 25 and 30 of the protocol (i.e. on day 4of the pre-hepatic phase and days 2, 9, 11 and 16 of the differentiationand maturation phase; FIG. 2 C), or long term/continuous treatment fromday 14 and onwards (i.e. starting on day 1 of the differentiation andmaturation phase; FIG. 2D).

Immediately after end of the RA treatment, the cell cultures aresubjected to a CYP activity assay according to the following protocol:Cells are washed twice with warm Williams medium E w/o phenol red (+0.1%PEST). Then CYP activity assay, consisting of warm Williams medium E w/ophenol red (+0.1% PEST), 2 mM L-Glutamine, 25 mM HEPES, 26 μM Phenacetin(model substrate for CYP1A), 9 μM Diclofenac (model substrate forCYP2C9) and 3 μM Midazolam (model substrate for CYP3A), is added to thecells (e.g. 220 μl/24 well) and incubated for 16 hr at 37° C. Thensupernatant is collected and centrifuged for 20 min at 10.000 rcf at 4°C. Subsequently, 120 μl of the supernatant is transferred into a 96 wellplate which is sealed with a tight seal tape and stored at −20 or −80°C. until LC/MS-analysis of metabolite formation: Acetaminophen(Paracetamol) for CYP1A, OH-Diclofenac for CYP2C9 and OH-Midazolam forCYP3A.

Results:

A) After 5 and 24 hr RA-exposures on day 21 of the protocol (i.e. on day7 of the differentiation and maturation phase), an immediate increase ofCYP1A and 3A activity in hESC-derived hepatocytes can be observed: CYP1Aactivity is on same level as in primary hepatocytes cultured for 48 hr,whereas CYP3A activity is roughly 25% of primary hepatocytes culturedfor 48 hr. HepG2 have much lower CYP1A and 3A activity than hESC-derivedhepatocytes. On day 23 in the protocol no CYP2C9 activity could bedetected in hESC-derived hepatocytes.

B) 7 days after a single 5 hr RA-exposure on day 23 of the protocol(i.e. on day 9 of the differentiation and maturation phase) hESC-derivedhepatocytes still have increased CYP1A, 2C9 and 3A activities comparedwith untreated cells. However the increase in CYP1A and 2C9 activity ishigher after 5 repeated 5 hr exposures (see FIG. 2C).

C) 5 repeated 5 hr RA pulses on days 11, 16, 23, 25 and 30 of theprotocol (i.e. on day 4 of the hepatic progenitor step and days 2, 9, 11and 16 of the maturation step) lead to a significant increase of CYP1A-,2C9- and 3A-activity on day 31 of the protocol. However, on day 24 ofthe protocol an increase of CYP1A-, but not of CYP3A-activity could beobserved (no CYP2C9-activity detectable on day 24 of the protocol).Thus, repeated 5 hr RA-exposures have a stronger increasing effect onCYP1A and 2C9 activity than one single 5 hr RA-exposure (comp. to FIG.2B).

D) A comparison of 5, 24, 48 hr exposures and continuous treatment showsthat the strongest increase of CYP3A activity is obtained withcontinuous RA-treatment compared to 5, 24 and 48 hr RA-exposures whereasthe strongest increase of CYP1A activity is observed after a 24 hrRA-exposure.

The strongest increase in expression of CYP2C9 is observed with repeated5 hr exposure commencing on d24 (i.e. day 12 of the differentiation andmaturation phase). Therefore the skilled person wishing to effect anincrease in a particular CYP gene may select from these pulse conditionsaccording to their gene of interest.

Example 5: Treatment with 9 Cis-Retinoic Acid (RA) Induces a More AdultPhenotype in hESC-Derived Hepatocyte-Like Cells Procedure:

Following the basic protocol A, hES cell derived hepatocyte-like cellscultured on a Gelatin-based coating were treated with 1 μM 9-cisretinoic acid for 5 hr on day 21 of the protocol (i.e. on day 7 of thedifferentiation and maturation phase) or 24 hr on day 22-23 of theprotocol (i.e. on day 8-9 of the differentiation and maturation phase).

Cells were harvested on day 21 or 23 of the protocol (i.e. on day 7 or 9of the differentiation and maturation phase) and gene expression wasanalysed using qRT-PCR, normalised to the house-keeping gene CREBBP andthe results presented as relative quantification normalised to acalibrator (FIG. 3.)

Results:

A) An increase of mRNA expression of the adult hepatic gene CYP3A4 isobserved immediately after a 5 RA-exposure on day 21 (i.e. on day 7 ofthe differentiation and maturation phase) as well as 2 and 7 days later(on day 23 and 30 of the protocol, respectively), Also a 24 hrRA-exposure on day 22-23 of the protocol (i.e. on day 8-9 of thedifferentiation and maturation phase) lead to an immediate up-regulationof adult CYP3A4 expression on day 23 (i.e. on day 9 of thedifferentiation and maturation phase).

B) A 5 hr exposure on day 21 (i.e. on day 7 of the differentiation andmaturation phase) decreases expression of the fetal hepatic gene CYP3A7slightly immediately after the exposure on day 21 and strongly 2 dayslater on day 23 of the protocol (i.e. on day 9 of the differentiationand maturation phase). Similarly, a 24 hr RA-exposure on day 22-23 ofthe protocol (i.e. on day 8-9 of the differentiation and maturationphase) strongly decreases expression of the fetal hepatic gene CYP3A7immediately after the exposure on day 23 of the protocol (i.e. on day 9of the differentiation and maturation phase).

C) A 5 hr exposure on day 21 (i.e. on day 7 of the differentiation andmaturation phase) increases mRNA expression of the adult hepatic genePXR on day 23 (i.e. on day 9 of the differentiation and maturationphase), but not immediately after the 5 hr exposure on day 21 (i.e. onday 7 of the differentiation and maturation phase).

A 24 hr RA-exposure on day 22-23 of the protocol (i.e. on day 8-9 of thedifferentiation and maturation phase) also increases mRNA expression ofthe adult hepatic gene PXR expression.

D,E) Continuous/long-term RA treatment leads to higher increase of mRNAexpression of the adult hepatic gene CAR (D) and a strongerdown-regulation of the fetal hepatic gene α-Fetoprotein (AFP, E) than 5and 24 hr RA exposures on d22 and day 21-22, respectively (day 8 and day7-8 of the differentiation and maturation phase, respectively).

In this case it can be seen that exposure to RA at d21 (at the end ofthe hepatic progenitor phase) leads to an increase in the expression ofadult genes CYP3A4, CAR and PXR and a decrease in fetal genes AFP andCYP3A7, thus showing that a more mature and adult phenotype is achieved.The skilled person can further refine this method by selecting 5 hrpulse, 24 hours pulse or continuous treatment if there is one specificgene or group of genes from within this set which they wish to up ordown regulate.

Example 6: Effect of Treatment of hESC- and hiPSC-DerivedHepatocyte-Like Cells with 9 Cis-Retinoic Acid (RA), Kenpaullone (K) anda Thin Fibronectin-Collagen I-Overlay (thinFC-Overlay) on CYP ActivityProcedure:

Following the basic protocol B, differentiating hES cell derived hepaticprogenitor cells and hepatocyte-like cells cultured on aFibronectin-based coating were treated with continuous/long termtreatment with 0.2 μM 9cis-retinoic acid and 0.5 μM Kenpaullone startingon day 14 of the protocol (i.e. day 1 of the differentiation andmaturation phase) and received thin Fibronectin-Collagen I-overlays onday 14 and 16 of the protocol (i.e. day 1 and 3 of the differentiationand maturation phase) and is refreshed thereafter once a week on day 23,30, 37 and so on (i.e. on day 9, 16, 23 and so on of the differentiationand maturation phase). This combination of thin Fibronectin-CollagenI-overlay, RA and Kenpaullone is called henceforth “RA+matrixoverlay+Kenpaullone”.

The thin Fibronectin-Collagen I-overlay is applied as following: Preparea 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stockwith 0.02M acetic acid. Pre-warm the cell culture medium to roomtemperature and add 8 μl of the 3 mg/ml Collagen I-solution per mlmedium (=25 μg Collagen I/ml) and 50 μl of a 1 mg/ml Fibronectinsolution per ml medium (=50 μg Fibronectin/ml). Remove the old mediumfrom the cultures and add 0.5 ml of the Collagen I andFibronectin-containing medium per cm² culture surface (=12.5 μg CollagenI/cm² and 25 μg Fibronectin/cm²). For refreshing the overlay once aweek, add 4 μl of the 3 mg/ml Collagen I-solution per ml medium (=6.25μg/ml) and 10 μl of a 1 mg/ml Fibronectin solution per ml medium (=5μg/ml).

For analysing functional expression of CYP enzymes, the cell culturesare subjected to a CYP activity assay according to the followingprotocol: Cells are washed twice with warm Williams medium E w/o phenolred (+0.1% PEST). Then CYP activity assay, consisting of warm Williamsmedium E w/o phenol red (+0.1% PEST), 2 mM L-Glutamine, 25 mM HEPES, 26μM Phenacetin (model substrate for CYP1A), 9 μM Diclofenac (modelsubstrate for CYP2C9) and 3 μM Midazolam (model substrate for CYP3A), isadded to the cells (e.g. 220 μl/24 well) and incubated for 16 hr at 37°C. Then supernatant is collected and centrifuged for 20 min at 10.000rcf at 4° C. Subsequently, 120 μl of the supernatant is transferred intoa 96 well plate which is sealed with a tight seal tape and stored at −20or −80° C. until LC/MS-analysis of metabolite formation: Acetaminophen(Paracetamol) for CYP1A, OH-Diclofenac for CYP2C9 and OH-Midazolam forCYP3A,

Results:

The inventors have found that, further to the use of RA alone, thecombination of continuous/long term treatment with a thinFibronectin-Collagen I-overlay, 0.5 μM Kenpaullone, and 0.2 μM RA[henceforth “RA+matrix overlay+Kenpaullone] (starting on day 14 andonwards, i.e. starting on day 1 of the differentiation and maturationphase) reproducibly increases CYP activity in hESC-derived hepatocytes(FIG. 4 A, B) and hiPSC-derived hepatocytes (FIG. 4 C) and thus inducesa more adult hepatocyte phenotype (more similar to human primaryhepatocytes)

A) The combination of continuous treatment of hESC-derived hepatocytestreated with 0.2 μM RA and 0.5 μM Kenpaullone (starting on day 14 andonwards) and the application of a thin Fibronectin-Collagen I-overlay isthe only experimental group which has both increased CYP2C9- and3A-activity on day 36 of the protocol (i.e. day 22 of thedifferentiation and maturation phase). However, some single or doubletreated groups also showed increase of CYP2C9- and 3A-activity, but nonehad both high CYP2C9- and 3A-activity. CYP1A activity is highest withoverlay alone. HepG2 only show CYP1A activity and no 2C9 or 3A activity.

B,C) The combination of continuous/long term treatment with a thinFibronectin-Collagen I-overlay, 0.5 μM Kenpaullone, and 0.2 μM RA(starting on day 14 and onwards) reproducibly increases CYP1A, 2C9 and3A activity in hESC-derived and hiPSC-derived hepatocytes.

The expression levels of various CYP genes and other markers associatedwith a mature hepatic phenotype have been further examined in thefollowing examples, which provide more detailed guidance for thosewishing to improve mature hepatic phenotype using this triplecombination.

Example 7: Increase in Expression of Hepatic Phase I and Phase IIEnzymes, Drug Transporters and Nuclear Receptors in Hepatocyte-LikeCells Procedure:

Following the basic protocols B (FIG. 5), C (hESC-derived hepatocytes inFIG. 6) or D (hiPSC-derived hepatocytes in FIG. 6), differentiating hPScell derived hepatic progenitor cells and hepatocyte-like cells culturedon a Fibronectin-based coating were treated with continuous/long termtreatment with 0.2 μM 9cis-RA and 0.5 μM Kenpaullone starting on day 14of the protocol (i.e. on day 1 of the differentiation and maturationphase) and received thin Fibronectin-Collagen I-overlays on day 14 and16 of the protocol (i.e. on day 1 and 3 of the differentiation andmaturation phase) and is refreshed thereafter once a week on day 23, 30,37 and so on (i.e. on day 9, 16, 23 and so on of the differentiation andmaturation phase).

The thin Fibronectin-Collagen I-overlay is applied as following: Preparea 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stockwith 0.02M acetic acid. Pre-warm the cell culture medium to roomtemperature and add 8 μl of the 3 mg/ml Collagen I-solution per mlmedium (=25 μg Collagen I/ml) and 50 μl of a 1 mg/ml Fibronectinsolution per ml medium (=50 μg Fibronectin/ml). Remove the old mediumfrom the cultures and add 0.5 ml of the Collagen I andFibronectin-containing medium per cm² culture surface (=12.5 μg CollagenI/cm² and 25 μg Fibronectin/cm²). For refreshing the overlay once aweek, add 4 μl of the 3 mg/ml Collagen I-solution per ml medium (=6.25μg/ml) and 10 μl of a 1 mg/ml Fibronectin solution per ml medium (=5μg/ml).

Cells were harvested on day 23, 30 and 36/37 of the protocol (i.e. onday 9, 16 and 22/23 of the differentiation and maturation phase) andgene expression was analysed using qRT-PCR, normalised to thehouse-keeping gene CREBBP, and the results presented as relativequantification normalised to a calibrator (FIGS. 5 and 6).

Results:

FIGS. 5 and 6 summarise results from several experiments where severalhESC lines (FIG. 5) or hESC and hiPSC (FIG. 6) from several independentlines were used to generate hepatocyte-like cells. Those were thenexposed to a combination of retinoic acid, Kenpaullone and matrixoverlay before being assayed by QRT-PCR for mRNA expression of a numberof markers for mature hepatocytes including NTCP, GSTA1-1, CAR, CYP2B6,CYP2C9, CYP3A4, CYP3A5, CYP1A2, CYP2D6.

FIG. 5 A-F) Upon treatment with a combination of RA, Kenpaullone andmatrix overlay, hepatocyte-like cells derived from three differenthESC-lines (SA181, SA167 and SA461; using basic protocol B) showedsimilar tendencies of increased mRNA expression of the adult hepaticmarkers NTCP, GSTA1-1, CAR, CYP2B6, CYP2C9, and CYP3A4 on day 23, 30 and37 of the protocol (i.e. on day 9, 16 and 23 of the differentiation andmaturation phase).

FIG. 6 A-G) Upon treatment with a combination of RA, Kenpaullone andmatrix overlay, both hepatocyte-like cells derived from hESC and hiPSC(using basic protocols C and D, respectively) showed similar increasesof mRNA expression of the adult hepatic markers CYP2B6, CYP3A4, CYP3A5,CAR, GSTA1-1, NTCP and CYP1A2 on day 29 and 36 of the protocol (i.e. onday 15 and 22 of the differentiation and maturation phase).

The combination of Kenpaullone, matrix overlay and RA shows asynergistic effect over exposures with RA alone (see e.g. CYP3A4 mRNAexpression in FIG. 3A versus FIG. 5F, or CYP2C9 and 3A activity in FIG.4A). The effect is consistent across several independent hESC lines andacross an hiPS cell line, as illustrated in the paired images of FIG. 6which compares gene expression in hESC- and hiPS-derived hepatocyte-likecells for a number of genes.

The skilled person may therefore select from a number of sources ofpluripotent stem cell lines as starting material to implement theinvention. The skilled person may also employ the results obtained inFIGS. 3, 5 and 6 to selectively upregulate one or more gene markers byselecting a treatment (RA exposure alone, or RA+matrix overlay+GSK-3inhibitor or CDK inhibitor) and a specified time point(s) for treatmentaccording to which markers they wish to upregulate.

Example 8: Increase in Expression of Hepatic Phase I and Phase IIEnzymes, Drug Transporters and Nuclear Receptors in Hepatocyte-LikeCells: Pre-Exposure to DNA Demethylation Agent Procedure:

Following the basic protocols C (hESC-derived hepatocytes) or D(hiPSC-derived hepatocytes), differentiating hES and hiPS cells in thepre-endodermal phase were treated with 10 nM of the DNA demethylatingagent 5-aza-2-deoxycytidine for 24 hr on day 2 to 3 of the protocol.

Later in the protocol, hPS cell derived hepatic progenitor cells andhepatocyte-like cells cultured on a Fibronectin-based coating weretreated with continuous/long term treatment with 0.2 μM 9cis-RA and 0.5μM Kenpaullone starting on day 14 of the protocol (i.e. on day 1 of thedifferentiation and maturation phase) and received thinFibronectin-Collagen I-overlays on day 14 and 16 of the protocol (i.e.on day 1 and 3 of the differentiation and maturation phase) and isrefreshed thereafter once a week on day 23, 30, 37 and so on (i.e. onday 9, 16, 23 and so on of the differentiation and maturation phase).

The thin Fibronectin-Collagen I-overlay is applied as following: Preparea 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stockwith 0.02M acetic acid. Pre-warm the cell culture medium to roomtemperature and add 8 μl of the 3 mg/ml Collagen I-solution per mlmedium (=25 μg Collagen I/ml) and 50 μl of a 1 mg/ml Fibronectinsolution per ml medium (=50 μg Fibronectin/ml). Remove the old mediumfrom the cultures and add 0.5 ml of the Collagen I andFibronectin-containing medium per cm² culture surface (=12.5 μg CollagenI/cm² and 25 μg Fibronectin/cm²). For refreshing the overlay once aweek, add 4 μl of the 3 mg/ml Collagen I-solution per ml medium (=6.25μg/ml) and 10 μl of a 1 mg/ml Fibronectin solution per ml medium (=5μg/ml).

For analysis of mRNA expression (FIG. 9), cells were harvested on day 29and 36 of the protocol (i.e. on day 15 and 22 of the differentiation andmaturation phase) and gene expression was analysed using qRT-PCR,normalised to the house-keeping gene CREBBP, and the results presentedas relative quantification normalised to a calibrator.

For analysing functional expression of CYP enzymes on day 36 (i.e. onday 22 of the maturation step; FIG. 10), the cell cultures are subjectedto a CYP activity assay according to the following protocol: Cells arewashed twice with warm Williams medium E w/o phenol red (+0.1% PEST).Then CYP activity assay, consisting of warm Williams medium E w/o phenolred (+0.1% PEST), 2 mM L-Glutamine, 25 mM HEPES, 26 μM Phenacetin (modelsubstrate for CYP1A), 9 μM Diclofenac (model substrate for CYP2C9) and 3μM Midazolam (model substrate for CYP3A), is added to the cells (e.g.220 μl/24 well) and incubated for 16 hr at 37° C. Then supernatant iscollected and centrifuged for 20 min at 10.000 rcf at 4° C.Subsequently, 120 μl of the supernatant is transferred into a 96 wellplate which is sealed with a tight seal tape and stored at −20 or −80°C. until LC/MS-analysis of metabolite formation: Acetaminophen(Paracetamol) for CYP1A, OH-Diclofenac for CYP2C9 and OH-Midazolam forCYP3A,

Results:

FIG. 9 A-G: Upon treatment with the demethylating agent5-aza-2-deoxycytidine during the pre-endodermal phase and a combinationof RA, Kenpaullone and matrix overlay during the differentiation andmaturation phase, both hepatocyte-like cells derived from hESC and hiPSCshowed similar increases of mRNA expression of the adult hepatic markersCYP2B6, CYP3A4, CYP3A5, CAR, GSTA1-1, NTCP and CYP1A2 on day 29 and 36of the protocol (i.e. on day 15 and 22 of the differentiation andmaturation phase).

FIG. 10 A,B: Treatment with a combination of RA, Kenpaullone and matrixoverlay during the differentiation and maturation phase reproduciblyincreases CYP1A, 2C9 and 3A activity in hESC-derived and hiPSC-derivedhepatocytes which were derived from hPS cells treated with thedemethylating agent 5-aza-2-deoxycytidine during the pre-endodermalphase.

FIG. 11: Corresponding morphology of cells can be seen in FIG. 11 whichdisplays cell morphology of hESC- and hiPSC-derived hepatocyte-likecells where differentiating hPS cells were treated with DNAdemethylation agent during pre-endodermal phase and obtainedhepatocyte-like cells then exposed to matrix overlay in combination withKenpaullone and RA. Images show that hepatic morphology is maintainedfor up to and beyond 42 days after initiation of hPS celldifferentiation in the hepatocyte-like cells obtained by cell treatmentwith DNA demethylation agent, matrix overlay in combination withKenpaullone and RA whereas untreated control cells start to die off orde-differentiate

FIG. 12 A,B: Here a comparison of CYP activity in hESC- andhiPSC-derived hepatocytes obtained with or without treatment with thedemethylating agent 5-aza-2-deoxycytidine on day 2-3 of the protocol andwith or without continuous treatment with 0.2 μM 9cis-RA and 0.5 μMKenpaullone during the differentiation and maturation phase ispresented. For both CYP1A and 3A activity the highest values areobtained for hepatocyte-like cells treated with all 4 factors,5-aza-2-deoxycytidine, matrix overlay, Kenpaullone and RA, suggesting asynergistic effect of those 4 factors on CYP1A and 3A activity and theinduction of the most mature hepatic phenotype by combined treatmentwith all 4 factors. No additional increase on CYP2C9 activity could beobserved due to treatment with a DNA demethylation agent.

The general trend seen here is that both hiPS and hESC-derivedhepatocytes show increase in expression of mature markers; withtreatment generating a small increase initially (d29) and a largerincrease by d36. For example, FIG. 9A shows that the combination ofDNA-demethylation treatment combined with later RA+matrixoverlay+Kenpaullone gives an increase in expression of CYP2B6 in bothhESC and iPS-derived hepatocyte-like cells, and that this increase ismore marked at d36 than d29. The synergistic effect of combining earlyDNA-demethylation treatment with later RA+matrix overlay+Kenpaullone canbe illustrated in, for example, the expression of NTCP (see FIG. 5Aversus FIG. 9F) where higher fold NTCP expression is the result of thissynergy. Once again, the skilled person may utilise the resultsexemplified in FIGS. 9 and 10 to improve overall hepatic phenotype oftreated cells or to upregulate one or more selected gene markers.

Example 9: Stable CYP Expression in Hepatocyte-Like Cells Derived fromhESC and hiPSC Treated with a Demethylating Agent, Retinoic Acid,Kenpaullone and a Matrix Overlay

Procedure:

Following the basic protocols C (hESC-derived hepatocytes) or D(hiPSC-derived hepatocytes), cells in the DE-step were treated with 10nM of the DNA demethylating agent 5-aza-2-deoxycytidine for 24 hr on day2 to 3 of the protocol. Later in the protocol, hPS cell derived hepaticprogenitor cells and hepatocyte-like cells cultured on aFibronectin-based coating were treated with continuous/long termtreatment with 0.2 μM 9cis-RA and 0.5 μM Kenpaullone starting on day 14of the protocol (i.e. on day 1 of the maturation step) and received thinFibronectin-Collagen I-overlays on day 14 and 16 of the protocol (i.e.on day 1 and 3 of the differentiation and maturation phase) and isrefreshed thereafter once a week on day 23, 30, 37 and so on (i.e. onday 9, 16, 23 and so on of the differentiation and maturation phase).

The thin Fibronectin-Collagen I-overlay is applied as following: Preparea 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stockwith 0.02M acetic acid. Pre-warm the cell culture medium to roomtemperature and add 8 μl of the 3 mg/ml Collagen I-solution per mlmedium (=25 μg Collagen I/ml) and 50 μl of a 1 mg/ml Fibronectinsolution per ml medium (=50 μg Fibronectin/ml). Remove the old mediumfrom the cultures and add 0.5 ml of the Collagen I andFibronectin-containing medium per cm² culture surface (=12.5 μg CollagenI/cm² and 25 μg Fibronectin/cm²). For refreshing the overlay once aweek, add 4 μl of the 3 mg/ml Collagen I-solution per ml medium (=6.25μg/ml) and 10 μl of a 1 mg/ml Fibronectin solution per ml medium (=5μg/ml).

For analysis of mRNA expression (FIG. 9), hESC- and hiPSC-derivedhepatocytes were harvested on day 22, 29 and 36 of the protocol (i.e. onday 8, 15 and 22 of the maturation step) and human primary hepatocytes 4and 48 hr after plating. Gene expression was analysed using qRT-PCR,normalised to the house-keeping gene CREBBP, and the results presentedas relative quantification normalised to a calibrator.

For analysing CYP enzyme activity in hESC- and hiPSC-derived hepatocyteson day 22, 29 and 36 (i.e. on day 8, 15 and 22 of the maturation step)and human primary hepatocytes 4, 24, 48, 72 and 96 hr after plating,cell cultures were subjected to a CYP activity assay according to thefollowing protocol: Cells are washed twice with warm Williams medium Ew/o phenol red (+0.1% PEST). Then CYP activity assay, consisting of warmWilliams medium E w/o phenol red (+0.1% PEST), 2 mM L-Glutamine, 25 mMHEPES, 26 μM Phenacetin (model substrate for CYP1A), 9 μM Diclofenac(model substrate for CYP2C9) and 3 μM Midazolam (model substrate forCYP3A), is added to the cells (e.g. 220 μl/24 well) and incubated for 16hr at 37° C. Then supernatant is collected and centrifuged for 20 min at10.000 rcf at 4° C. Subsequently, 120 μl of the supernatant istransferred into a 96 well plate which is sealed with a tight seal tapeand stored at −20 or −80° C. until LC/MS-analysis of metaboliteformation: Acetaminophen (Paracetamol) for CYP1A, OH-Diclofenac forCYP2C9 and OH-Midazolam for CYP3A,

Results:

The inventors have further investigated the long-term effects of earlyDNA-demethylation treatment combined with late RA+matrixoverlay+Kenpaullone to determine whether hepatic phenotype andexpression of hepatic markers genes is maintained after long periods inculture.

FIG. 16 A,B: HESC and hiPSC-derived hepatocyte-like cells obtained bytreatment with a DNA demethylating agent during early endodermaldevelopment and exposure to retinoic acid, Kenpaullone and matrixoverlay during the differentiation and maturation phase show asurprisingly stable or increasing level of CYP1A, 2C9 and 3A activity(FIG. 16 A-2) as well as a stable or increasing mRNA expression ofseveral CYPs (FIG. 16 B) in contrast to human primary hepatocytes whichtypically quickly lose CYP activity and mRNA expression in culture.HepG2 have very low or no expression of many adult hepatic genes. Thusthe skilled person may employ this treatment technique and be assuredthat long-term maintenance of hepatic phenotype is possible; they mayfurther tailor a treatment programme based on this and on previousexamples should they wish to generate long-term expression of just oneor more specific markers.

Example 10: Validation of Improved Definitive Endoderm Phenotype inhESC- and hiPSC-Derived DE Treated with a DNA Demethylation AgentProcedure:

Following the basic protocol C (both for hESC- and hiPSC-derivedhepatocytes), cells were treated with 10 nM 5-aza-2-deoxycytidine atdifferent time points and for different durations during thepre-endodermal phase, e.g. on day 2-3, 2-4, 3-4 and 4-6 of the protocol(hESC-DE: no 5azadC n=4, 5azadC d2-3 n=4, d3-4 n=1, d2-4 n=1, d4-6 n=1;hiPSC-DE: no 5azadC n=5, 5azadC d2-3 n=5, d3-4 n=2, d2-4 n=1, d4-6 n=1;with n being the number of individual experiments).

For analysis of mRNA expression, hESC- and hiPSC-derived DE-cells wereharvested on day 7 of the protocol and gene expression was analysedusing qRT-PCR, normalised to the house-keeping gene CREBBP, and theresults presented as relative quantification normalised to a calibrator.

Results: FIG. 13A:

DE derived from hESC treated with 10 nM 5-aza-2-deoxycytidine on day 2-3(FIG. 13 A2) is more homogeneous and has more pronounced cell-cellcontacts compared to untreated control DE (FIG. 13 A1). Note thepresence of undifferentiated cells in the control DE (FIG. 13 A1) whichis in accordance with higher expression of Oct4 and Nanog mRNAexpression in control DE (compare FIG. 13 D). Similar results wereobtained when treating cells on days 2-4, 3-4 and 4-6 and with 100 nM5-aza-2-deoxycytidine. 1 nM 5-aza-2-deoxycytidine had less effect (datanot shown).

FIG. 13B:

HiPSC-derived DE treated with 10 nM 5-aza-2-deoxycytidine on day 2-3(FIG. 13 B2) is more confluent and has more pronounced cell-cellcontacts than control DE (FIG. 13 B1). Similar results were obtainedwhen treating cells days 2-4, 3-4 and 4-6 and with 100 nM5-aza-2-deoxycytidine. 1 nM 5-aza-2-deoxycytidine had less effect (datanot shown).

FIG. 13C:

HiPSC-derived DE treated with 10 nM 5-aza-2-deoxycytidine on day 2-3 hasmuch less Oct4-immunopositive cells at day 7 compared to untreatedcontrols, i.e. less undifferentiated cells are left and the DE is morehomogeneous after treatment with a demethylating agent.

FIG. 13D:

Expression of the stem cell marker Oct4 is much lower in hESC- andhiPSC-derived DE treated with 10 nM 5azadC on day 2-3, 3-4, 2-4, and 4-6than in untreated controls (FIG. 13 D1). In 5azadC-treated hESC-derivedDE mRNA expression of the stem cell marker Nanog is strongly decreasedwhereas it remains mainly unaffected in hiPSC-derived DE (FIG. 13 D1).Expression of the DE markers Sox17, Cxcr4, FoxA2 and hHex isup-regulated in 5azadC-treated hESC- and hiPSC-derived DE compared tountreated controls while the effect is stronger in hESC-derived DE thanin hiPSC-derived DE (FIG. 13 D3-6). Expression of the extraembryonicmarker Sox7 is very low both in control and 5azadC-treated hESC- andhiPSC-derived DE with the exception of 5azadC-treatment on days 2-4 and4-6 which increases Sox7 mRNA levels.

Taken together, the treatment of the cells with a DNA demethylationagent during the pre-endodermal phase led to improved DE morphology andDE cell yield in both hESC and hiPSC derived cells (FIG. 13 A-B).Furthermore it resulted in a stronger decrease of the stem cell markerOct4 as detected by immunocytochemistry (FIG. 13 C), to an improvedexpression of well defined DE markers SOX17, CXCR4, HEX, Foxa2, as wellas a decrease of the extraembryonic endoderm marker Sox7 and of the stemmarkers Oct4 and Nanog (FIG. 13 D). Therefore the skilled person wishingto produce a more homogeneous population of definitive endoderm cellscan select from one or more DNA-demethylation agents and employ theme.g. at days 2-3 or 3-4 during differentiation of pluripotent stem celltypes.

Example 11: Highly Homogeneous Definitive Endoderm Derived from a Panelof 27 hPSC Lines Upon Treatment with a DNA Demethylating Agent During DEDifferentiation Procedure:

Following the basic protocols C or D, cells derived from 27 hPSC lineswere treated with 10 nM 5-aza-2-deoxycytidine on day 2-3 during thepre-endodermal phase (protocol C: ChiPSC14, ChiPSC19, ChiPSC22, P11015,SA167, SA181, SA461, and Val9; protocol D: ChiPSC4, ChiPSC6b, ChiPSC7,ChiPSC8, ChiPSC9, ChiPSC10, ChiPSC11, ChiPSC13, ChiPSC15, ChiPSC17,ChiPSC18, ChiPSC19, ChiPSC20, ChiPSC21, ChiPSC23, ChiPSC24, P11012,P11021, P11025, and SA121).

23 out of 27 hPSC lines were tested with both protocols C and D. Out ofthese 23 lines, only 4 cell lines (ChiPSC14, ChiPSC23, P11015, andP11032) could only be differentiated with one of the two protocols. FourhPSC lines (ChiPSC8, ChiPSC9, ChiPSC10, and ChiPSC11) were only testedwith protocol D.

For analysis of mRNA expression, hESC- and hiPSC-derived DE-cells wereharvested on day 7 of the protocol and gene expression was analysedusing qRT-PCR, normalised to the house-keeping gene CREBBP, and theresults presented as relative quantification normalised to a calibrator.

Results: FIG. 14A-D:

Using the basic protocols C or D including a DNA demethylating treatmenton day 2-3 during the pre-endodermal phase, undifferentiated stem cellsfrom 27 different hPSC lines could be differentiated into highlyhomogeneous DE displaying low mRNA expression levels of the stem cellmarkers Oct-4 and Nanog (FIG. 14A, B) and high levels of the DE markersSox17 and Cxcr4 (FIG. 14C, D) compared to undifferentiated hESC (SA181)and hiPSC (ChiPSC4).

Taken together, the treatment of the cells during the pre-endodermalphase with a DNA demethylating agent allows derivation of homogeneous DEwith low expression levels of stem cell markers and high expressionlevels of DE markers from all hPSC lines tested. The derivation ofhomogeneous DE is crucial for derivation of homogeneous hepatocytecultures which could be obtained from all lines tested (data not shown).

Therefore the skilled person wishing to produce a homogeneous populationof definitive endoderm cells (and hepatocytes) from any given hPSC linecan include a treatment with a DNA demethylating agent, for instance, onday 2-3 during the pre-endodermal phase.

Example 12: Both DNA Demethylating Agents 5-Aza-2-Deoxycytidine and5-Azacytidine Improve the Definitive Endoderm Phenotype in hESC- andhiPSC-Derived DE Procedure:

Following the basic protocols C (P11032, SA181) or D (P11012), cellsderived from 3 different hPSC lines were treated with either 10 nM5-aza-2-deoxycytidine or 1 μM 5-azacytidine on day 2-3 during thepre-endodermal phase.

For analysis of mRNA expression, hESC- and hiPSC-derived DE-cells wereharvested on day 7 of the protocol and gene expression was analysedusing qRT-PCR, normalised to the house-keeping gene CREBBP, and theresults presented as relative quantification normalised to a calibrator.

Results: FIG. 15:

A, B) Without treatment with a demethylating agent, the three hPSC linesP11032, SA181 and P11012 produced heterogeneous DE with relatively highmRNA expression of stem cell markers Oct4 and Nanog (FIG. 15A, B).Treatment with the DNA demethylating agents 5-aza-2-deoxycytidine(5azadC) and 5-azacytidine (5azaC) significantly decreased Oct4 andNanog mRNA (FIG. 15 A, B) and thus allowed derivation of a homogeneousDE population from these three hPSC lines.

C, D) No significant changes in mRNA expression of the DE markers Sox17and Cxcr4 could be observed upon treatment with 10 nM5-aza-2-deoxycytidine or 1 μM 5-azacytidine (FIG. 15 C, D).

Taken together, treatment with both DNA demethylating agents5-aza-2-deoxycytidine (5azadC) and 5-azacytidine (5azaC) allowsderivation of homogeneous DE from hPSC lines, giving otherwiseheterogeneous DE if untreated.

Therefore the skilled person wishing to produce a homogeneous populationof definitive endoderm cells can select from one or moreDNA-demethylation agents and employ them e.g. at days 2-3 duringdifferentiation of pluripotent stem cell types.

Example 13: Further Improvement of Functionality in Hepatocyte-LikeCells Derived from hESC and hiPSC Treated with a Demethylating Agent,Two Activators of a Retinoic Acid Responsive Receptor, Kenpaullone and aMatrix Overlay Procedure:

Following the basic protocol D (hiPSC-derived hepatocytes),differentiating hPS cells in the pre-endodermal phase were treated with10 nM of the DNA demethylating agent 5-aza-2-deoxycytidine for 24 hr onday 2 to 3 of the protocol. Later in the protocol, hPS cell derivedhepatic progenitor cells and hepatocyte-like cells cultured on aFibronectin-based coating were treated with continuous/long termtreatment with 0.2 μM 9cis-RA and 0.5 μM Kenpaullone starting on day 14of the protocol (i.e. on day 1 of the differentiation and maturationphase) and received thin Fibronectin-Collagen I-overlays on day 14 and16 of the protocol (i.e. on day 1 and 3 of the differentiation andmaturation phase) and which are refreshed thereafter once a week on day23, 30, 37 and so on (i.e. on day 9, 16, 23 and so on of thedifferentiation and maturation phase). One group received in addition tothe described treatment 0.2 μM 13cis-RA starting from day 21 of theprotocol (i.e. on day 7 of the differentiation and maturation phase).

The thin Fibronectin-Collagen I-overlay is applied as following: Preparea 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stockwith 0.02M acetic acid. Pre-warm the cell culture medium to roomtemperature and add 8 μl of the 3 mg/ml Collagen I-solution per mlmedium (=25 μg Collagen I/ml) and 50 μl of a 1 mg/ml Fibronectinsolution per ml medium (=50 μg Fibronectin/ml). Remove the old mediumfrom the cultures and add 0.5 ml of the Collagen I andFibronectin-containing medium per cm² culture surface (=12.5 μg CollagenI/cm² and 25 μg Fibronectin/cm²). For refreshing the overlay once aweek, add 4 μl of the 3 mg/ml Collagen I-solution per ml medium (=6.25μg/ml) and 10 μl of a 1 mg/ml Fibronectin solution per ml medium (=5μg/ml).

For analysis of mRNA expression, hESC- and hiPSC-derived hepatocyteswere harvested on day 36 of the protocol (i.e. on day 22 of thedifferentiation and maturation phase) and human primary hepatocytes 48hr after plating. Gene expression was analysed using qRT-PCR, normalisedto the house-keeping gene CREBBP, and the results presented as relativequantification normalised to a calibrator.

For analysing CYP enzyme activity in hiPSC-derived hepatocytes on day 36(i.e. on day 22 of the differentiation and maturation phase), HepG2 andhuman primary hepatocytes 48 hr after plating, cell cultures weresubjected to a CYP activity assay according to the following protocol:Cells are washed twice with warm Williams medium E w/o phenol red (+0.1%PEST). Then CYP activity assay, consisting of warm Williams medium E w/ophenol red (+0.1% PEST), 2 mM L-Glutamine, 25 mM HEPES, 10 μM Phenacetin(model substrate for CYP1A), 10 μM Bupropion (model substrate forCYP2B6), 10 μM Diclofenac (model substrate for CYP2C9), 10 μM Bufuralol(model substrate for CYP2D6) and 5 μM Midazolam (model substrate forCYP3A), is added to the cells (e.g. 220 μl/24 well) and incubated for 16hr at 37° C. Then supernatant is collected and centrifuged for 20 min at10.000 rcf at 4° C. Subsequently, 120 μl of the supernatant istransferred into a 96 well plate which is sealed with a tight seal tapeand stored at −20 or −80° C. until LC/MS-analysis of metaboliteformation: Acetaminophen (Paracetamol) for CYP1A, OH-Bupropion forCYP2B6, OH-Diclofenac for CYP2C9, OH-Bufuralol for CYP2D6, andOH-Midazolam for CYP3A,

Results:

The inventors have further investigated the effects of treatment with anadditional activator of a retinoic acid responsive receptor in additionto early DNA-demethylation treatment and with late RA+matrixoverlay+Kenpaullone treatment to determine if this further improvedhepatocyte maturation.

FIG. 17 A-E:

HiPSC-derived hepatocyte-like cells obtained by treatment with a DNAdemethylating agent during early endodermal development and exposure to9cis RA, Kenpaullone and matrix overlay during the differentiation andmaturation phase showed a further increase of CYP1A-, CYP2B6-, CYP2C9-,CYP2D6- and CYP3A-activity upon treatment with 13cis RA.

FIG. 18 A-E:

HiPSC-derived hepatocyte-like cells obtained by treatment with a DNAdemethylating agent during early endodermal development and exposure to9cis RA, Kenpaullone and matrix overlay together with the additionalactivator of a retinoic acid responsive receptor 13cis RA during thedifferentiation and maturation phase showed the highest mRNA expressionlevels of CYP2C9, CYP3A4, CYP3A5 and PXR.

In contrast to an increase of CYP2B6 activity (FIG. 17 B), a decrease ofCYP2B6 mRNA expression can be found (FIG. 18 A) in the 13cis RA-treatedgroup.

Thus the skilled person may employ treatment with more than oneactivator of a retinoic acid responsive receptor, e.g. with two, three,four or more activators, in order to obtain hepatocyte-like cells withthe most mature characteristics.

Example 14: Further Improvement of Functionality in Hepatocyte-LikeCells Derived from hESC and hiPSC Treated with a Demethylating Agent,Retinoic Acid, Kenpaullone and More Complex Matrix Overlay Procedure:

Following the basic protocols C (hESC-derived hepatocytes) and D(hiPSC-derived hepatocytes), cells in the pre-endodermal phase weretreated with 10 nM of the DNA demethylating agent 5-aza-2-deoxycytidinefor 24 hr on day 2 to 3 of the protocol.

Later in the protocol, hPS cell derived hepatic progenitor cells andhepatocyte-like cells derived from hPS cells were treated withcontinuous/long term treatment with 0.2 μM 9cis-RA and 0.5 μMKenpaullone starting on day 14 of the protocol (i.e. on day 1 of thedifferentiation and maturation phase) and received thinFibronectin-Collagen I-overlays on day 14 and 16 of the protocol (i.e.on day 1 and 3 of the differentiation and maturation phase) and whichare refreshed thereafter once a week on day 23, 30, 37 and so on (i.e.on day 9, 16, 23 and so on of the differentiation and maturation phase).

One experimental group was grown on a liver matrix like-coating,consisting of Fibronectin, Collagen I, Collagen IV, Laminin,Nidogen/Entactin and Biglycan, and received a thin liver matrixlike-overlay consisting of Fibronectin, Collagen I, Collagen IV,Laminin, Nidogen/Entactin and Biglycan.

Another experimental group was cultured on a Fibronectin-CollagenI-basal lamina mix-coating, consisting of Fibronectin, Collagen I and apreparation of human extracellular matrix (including collagens, laminin,fibronectin, tenascin, elastin, proteoglycans and glycosaminoglycans),and received a thin Fibronectin-Collagen I-basal lamina mix-overlay,consisting of Fibronectin, Collagen I and a preparation of humanextracellular matrix (including collagens, laminin, fibronectin,tenascin, elastin, proteoglycans and glycosaminoglycans).

The control group was grown on the standard Fibronectin-based coatingand received thin Fibronectin-Collagen I-overlays on day 14 and 16 ofthe protocol (i.e. on day 1 and 3 of the differentiation and maturationphase) and which are refreshed thereafter once a week on day 23, 30, 37and so on (i.e. on day 9, 16, 23 and so on of the differentiation andmaturation phase).

The thin Fibronectin-Collagen I-overlay is applied as following: Preparea 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stockwith 0.02M acetic acid. Pre-warm the cell culture medium to roomtemperature and add 8 μl of the 3 mg/ml Collagen I-solution per mlmedium (=25 μg Collagen I/ml) and 50 μl of a 1 mg/ml Fibronectinsolution per ml medium (=50 μg Fibronectin/ml). Remove the old mediumfrom the cultures and add 0.5 ml of the Collagen I andFibronectin-containing medium per cm² culture surface (=12.5 μg CollagenI/cm² and 25 μg Fibronectin/cm²). For refreshing the overlay once aweek, add 4 μl of the 3 mg/ml Collagen I-solution per ml medium (=6.25μg/ml) and 10 μl of a 1 mg/ml Fibronectin solution per ml medium (=5μg/ml).

For the thin liver matrix like-overlay, add 8 μl of the 3 mg/ml CollagenI-solution per ml medium (=25 μg Collagen I/ml), 50 μl of a 1 mg/mlFibronectin solution per ml medium (=50 μg Fibronectin/ml), 1.2 μl of a0.5 mg/ml Collagen IV-solution per ml medium (=0.6 μg/ml), 6 μl of a 100μg/ml Nidogen/Entactin-solution per ml medium (=0.6 μg/ml), 6 μl of a100 μg/ml Laminin1-solution per ml medium (=0.6 μg/ml), and 6 μl of a200 μg/ml Biglycan-solution per ml medium (=1.2 μg/ml). For the thinFibronectin-Collagen I-basal lamina-overlay, add 8 μl of the 3 mg/mlCollagen I-solution per ml medium (=25 μg Collagen I/ml), 50 μl of a 1mg/ml Fibronectin solution per ml medium (=50 μg Fibronectin/ml), and 6μl of a 1 mg/ml human extracellular matrix-preparation per ml medium (=6μg/ml), An example for a suitable human extracellular matrix preparationis MaxGel™ from Sigma-Aldrich.

For analysing CYP enzyme activity in hESC- and hiPSC-derived hepatocyteson day 36 (i.e. on day 22 of the differentiation and maturation phase),HepG2 and human primary hepatocytes 48 hr after plating, cell cultureswere subjected to a CYP activity assay according to the followingprotocol: Cells are washed twice with warm Williams medium E w/o phenolred (+0.1% PEST). Then CYP activity assay, consisting of warm Williamsmedium E w/o phenol red (+0.1% PEST), 2 mM L-Glutamine, 25 mM HEPES, 10μM Phenacetin (model substrate for CYP1A), 10 μM Bupropion (modelsubstrate for CYP2B6), 10 μM Diclofenac (model substrate for CYP2C9), 10μM Bufuralol (model substrate for CYP2D6) and 5 μM Midazolam (modelsubstrate for CYP3A), is added to the cells (e.g. 220 μl/24 well) andincubated for 16 hr at 37° C. Then supernatant is collected andcentrifuged for 20 min at 10.000 rcf at 4° C. Subsequently, 120 μl ofthe supernatant is transferred into a 96 well plate which is sealed witha tight seal tape and stored at −20 or −80° C. until LC/MS-analysis ofmetabolite formation: Acetaminophen (Paracetamol) for CYP1A,OH-Bupropion for CYP2B6, OH-Diclofenac for CYP2C9, OH-Bufuralol forCYP2D6, and OH-Midazolam for CYP3A,

Results:

The inventors have further investigated the effects of treatment withmore complex, ECM-like coatings and overlays in addition to earlyDNA-demethylation treatment and late retinoic acid and Kenpaullonetreatment to determine if this further improved hepatocyte maturationcompared to the standard Fibronectin-based coating and the thinFibronectin-Collagen I-overlay.

FIG. 19A-C:

HESC- and hiPSC-derived hepatocyte-like cells obtained by treatment witha DNA demethylating agent during early endodermal development andexposure to retinoic acid, Kenpaullone and a matrix overlay during thedifferentiation and maturation phase showed a higher CYP2C9-, andCYP3A-activity when both coating and overlay were more complex andECM-like compared to the standard Fibronectin-based coating and the thinFibronectin-Collagen I-overlay of the control group.

Thus the skilled person may employ treatment with more complex coatingsand overlays resembling the liver matrix in order to obtainhepatocyte-like cells with the most mature characteristics.

Example 15: Improvement of Functionality in Hepatocyte-Like CellsDerived from hiPSC Treated with a Demethylating Agent, Kenpaullone and 9Cis Retinoic Acid or Analogs of 9 Cis Retinoic Acid Procedure:

Following the basic protocol D, cells in the pre-endodermal phase weretreated with 10 nM of the DNA demethylating agent 5-aza-2-deoxycytidinefor 24 hr on day 2 to 3 of the protocol.

Later in the protocol, hPS cell derived hepatic progenitor cells andhepatocyte-like cells derived from hPS cells were treated withcontinuous/long term treatment with 0.2 μM 9cis-RA and 0.5 μMKenpaullone starting on day 14 of the protocol (i.e. on day 1 of thedifferentiation and maturation phase) and received thinFibronectin-Collagen I-overlays on day 14 and 16 of the protocol (i.e.on day 1 and 3 of the differentiation and maturation phase) and whichare refreshed thereafter once a week on day 23, 30, 37 and so on (i.e.on day 9, 16, 23 and so on of the differentiation and maturation phase).

Some experimental groups were treated with 0.5 μM all trans-retinoicacid (ATRA), 5 μM AM580, 0.2 μM 13cis-RA, 5 μM LGD1069, 5 μM LG100268 or5 μM SR11237 instead of 0.2 μM 9cis-RA.

The thin Fibronectin-Collagen I-overlay is applied as following: Preparea 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stockwith 0.02M acetic acid. Pre-warm the cell culture medium to roomtemperature and add 8 μl of the 3 mg/ml Collagen I-solution per mlmedium (=25 μg Collagen I/ml) and 50 μl of a 1 mg/ml Fibronectinsolution per ml medium (=50 μg Fibronectin/ml). Remove the old mediumfrom the cultures and add 0.5 ml of the Collagen I andFibronectin-containing medium per cm² culture surface (=12.5 μg CollagenI/cm² and 25 μg Fibronectin/cm²). For refreshing the overlay once aweek, add 4 μl of the 3 mg/ml Collagen I-solution per ml medium (=6.25μg/ml) and 10 μl of a 1 mg/ml Fibronectin solution per ml medium (=5μg/ml).

For analysing CYP enzyme activity in hESC-derived hepatocytes on day 36(i.e. on day 22 of the differentiation and maturation phase), cellcultures were subjected to a CYP activity assay according to thefollowing protocol: Cells are washed twice with warm Williams medium Ew/o phenol red (+0.1% PEST). Then CYP activity assay, consisting of warmWilliams medium E w/o phenol red (+0.1% PEST), 2 mM L-Glutamine, 25 mMHEPES, 10 μM Phenacetin (model substrate for CYP1A), 10 μM Bupropion(model substrate for CYP2B6), 10 μM Diclofenac (model substrate forCYP2C9), 10 μM Bufuralol (model substrate for CYP2D6) and 5 μM Midazolam(model substrate for CYP3A), is added to the cells (e.g. 220 μl/24 well)and incubated for 16 hr at 37° C. Then supernatant is collected andcentrifuged for 20 min at 10.000 rcf at 4° C. Subsequently, 120 μl ofthe supernatant is transferred into a 96 well plate which is sealed witha tight seal tape and stored at −20 or −80° C. until LC/MS-analysis ofmetabolite formation: Acetaminophen (Paracetamol) for CYP1A,OH-Bupropion for CYP2B6, OH-Diclofenac for CYP2C9, OH-Bufuralol forCYP2D6, and OH-Midazolam for CYP3A,

Results:

The inventors have investigated if other RXR- and RAR-agonist besides9cis-RA can induce an improved functionality of hiPSC-derivedhepatocytes.

FIG. 20:

Treatment with 13cis-RA increases CYP2C9 activity to similar levels asupon treatment with 9cis-RA whereas treatment with SR11237, ATRA, AM580,LGD1069 and LG100268 leads to a slightly smaller increase (FIG. 20).

Thus the skilled person may use other RXR- and RAR-agonist, e.g.13cis-RA, ATRA, AM580, LGD1069, LG100268 and SR11237, besides 9cis-RAfor obtaining more mature hepatocyte-like cells.

Example 16: Improvement of Functionality in Hepatocyte-Like CellsDerived from hESC and hiPSC Treated with a Demethylating Agent, 9 CisRetinoic and Kenpaullone Acid or Analogs of Kenpaullone Procedure:

Following the basic protocols C (hESC-derived hepatocytes) and D(hiPSC-derived hepatocytes), cells in the pre-endodermal phase weretreated with 10 nM of the DNA demethylating agent 5-aza-2-deoxycytidinefor 24 hr on day 2 to 3 of the protocol.

Later in the protocol, hPS cell derived hepatic progenitor cells andhepatocyte-like cells derived from hPS cells were treated withcontinuous/long term treatment with 0.2 μM 9cis-RA and 0.5 μMKenpaullone starting on day 14 of the protocol (i.e. on day 1 of thedifferentiation and maturation phase) and received thinFibronectin-Collagen I-overlays on day 14 and 16 of the protocol (i.e.on day 1 and 3 of the differentiation and maturation phase) and whichare refreshed thereafter once a week on day 23, 30, 37 and so on (i.e.on day 9, 16, 23 and so on of the differentiation and maturation phase).

One experimental group was treated with 0.5 μM Indirubin-3-oxime insteadof 0.5 μM Kenpaullone.

The thin Fibronectin-Collagen I-overlay is applied as following: Preparea 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stockwith 0.02M acetic acid. Pre-warm the cell culture medium to roomtemperature and add 8 μl of the 3 mg/ml Collagen I-solution per mlmedium (=25 μg Collagen I/ml) and 50 μl of a 1 mg/ml Fibronectinsolution per ml medium (=50 μg Fibronectin/ml). Remove the old mediumfrom the cultures and add 0.5 ml of the Collagen I andFibronectin-containing medium per cm² culture surface (=12.5 μg CollagenI/cm² and 25 μg Fibronectin/cm²). For refreshing the overlay once aweek, add 4 μl of the 3 mg/ml Collagen I-solution per ml medium (=6.25μg/ml) and 10 μl of a 1 mg/ml Fibronectin solution per ml medium (=5μg/ml).

For analysing CYP enzyme activity in hESC- and hiPSC-derived hepatocyteson day 36 (i.e. on day 22 of the differentiation and maturation phase),cell cultures were subjected to a CYP activity assay according to thefollowing protocol: Cells are washed twice with warm Williams medium Ew/o phenol red (+0.1% PEST). Then CYP activity assay, consisting of warmWilliams medium E w/o phenol red (+0.1% PEST), 2 mM L-Glutamine, 25 mMHEPES, 10 μM Phenacetin (model substrate for CYP1A), 10 μM Bupropion(model substrate for CYP2B6), 10 μM Diclofenac (model substrate forCYP2C9), 10 μM Bufuralol (model substrate for CYP2D6) and 5 μM Midazolam(model substrate for CYP3A), is added to the cells (e.g. 220 μl/24 well)and incubated for 16 hr at 37° C. Then supernatant is collected andcentrifuged for 20 min at 10.000 rcf at 4° C. Subsequently, 120 μl ofthe supernatant is transferred into a 96 well plate which is sealed witha tight seal tape and stored at −20 or −80° C. until LC/MS-analysis ofmetabolite formation: Acetaminophen (Paracetamol) for CYP1A,OH-Bupropion for CYP2B6, OH-Diclofenac for CYP2C9, OH-Bufuralol forCYP2D6, and OH-Midazolam for CYP3A,

Results:

The inventors have investigated if other CDK- and GSK3-inhibitorsbesides Kenpaullone can induce an improved functionality ofhiPSC-derived hepatocytes.

FIG. 21:

Treatment with Indirubin-3-oxime increases CYP2C9 and 3A activity tosimilar levels as upon treatment with Kenpaullone both in hiPSC-derivedhepatocytes (FIG. 21 A1, A2) and in hESC-derived hepatocytes (FIG. 21B1, B2).

Thus the skilled person may use other CDK- and GSK3-inhibitors besidesKenpaullone for obtaining more mature hepatocyte-like cells.

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1-88. (canceled)
 89. A composition comprising at least one activator of a retinoic acid responsive receptor and at least one selected from GSK3 inhibitor, activator of Wnt signalling and CDK inhibitor.
 90. A composition according to claim 89, comprising at least one activator of a retinoic acid responsive receptor and at least one GSK-3 inhibitor, and optionally at least one CDK inhibitor; or comprising at least one activator of a retinoic acid responsive receptor and at least one activator of Wnt signalling, and optionally at least one CDK inhibitor; or comprising at least one activator of a retinoic acid responsive receptor and at least one CDK inhibitor, and optionally at least one GSK3 inhibitor or at least one activator of Wnt signalling.
 91. The composition according to claim 89, comprising at least one activator of a retinoic acid responsive receptor and at least one GSK-3 inhibitor, and optionally at least one CDK inhibitor.
 92. The composition according to claim 91, wherein the activator of a retinoic acid responsive receptor is a retinoic acid.
 93. The composition according to claim 91, wherein the activator of a retinoic acid responsive receptor is a retinoic acid isomer, a retinoic acid analogue, or a retinoid.
 94. The composition according to claim 91, wherein the activator of a retinoic acid responsive receptor is 9-cis-retinoic acid, 13-cis-retinoic acid or a combination thereof.
 95. The composition according to claim 91, wherein the activator of a retinoic acid responsive receptor is 9-cis-retinoic acid.
 96. The composition according to claim 91, wherein the GSK3-inhibitor is selected from the group consisting of Kenpaullone, 1-Aza-Kenpaullone, Alsterpaullone, Aminopyrimidine CHIR99021 and Indirubin-3′-monoxime.
 97. The composition according to claim 91, wherein the GSK3-inhibitor is selected from the group consisting of Kenpaullone, 1-Aza-Kenpaullone, and Alsterpaullone.
 98. The composition according to claim 91, wherein the GSK3-inhibitor is Kenpaullone.
 99. The composition according to claim 91, wherein the at least one activator of a retinoic acid responsive receptor is selected from the group consisting of 9-cis-retinoic acid, 13-cis-retinoic acid or a combination thereof, and the at least one GSK-3 inhibitor is selected from the group consisting of Kenpaullone, 1-Aza-Kenpaullone and Alsterpaullone.
 100. The composition according to claim 91, comprising 9-cis-retinoic acid and Kenpaullone.
 101. The composition according to claim 89, wherein the activator of Wnt signalling is a Wnt protein.
 102. The composition according to claim 89, wherein the at least one CDK inhibitor is selected from the group consisting of Kenpaullone, 1-Aza-Kenpaullone, Alsterpaullone, Indirubin-3′-monoxime, SNS-032(BMS-387032), AT-7519 and AZD5438.
 103. A kit comprising at least one activator of a retinoic acid responsive receptor and at least one selected from GSK3 inhibitor, activator of Wnt signalling, and CDK inhibitor.
 104. The kit according to claim 103, comprising at least one activator of a retinoic acid responsive receptor, at least one GSK3 inhibitor, and optionally at least one extracellular matrix (ECM) component or ECM component mixture; or comprising at least one activator of a retinoic acid responsive receptor, at least one activator of Wnt signalling, and optionally at least one extracellular matrix (ECM) component or ECM component mixture; or comprising at least one activator of a retinoic acid responsive receptor, at least one CDK inhibitor, and optionally at least one extracellular matrix (ECM) component or ECM component mixture.
 105. The kit according to claim 103, comprising at least one activator of a retinoic acid responsive receptor, at least one GSK3 inhibitor, and optionally at least one extracellular matrix (ECM) component or ECM component mixture.
 106. The kit according to claim 103, wherein the kit comprises at least one extracellular matrix (ECM) component or ECM component mixture.
 107. The kit according to claim 106, wherein the at least one ECM component is selected from the group consisting of collagen, fibronectin, elastin, chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, heparin proteoglycan, heparan sulfate proteoglycan, such as glypicans, syndecans or perlecans, glycosaminoglycans, nidogen/entactin, laminins, biglycan, tenascin, and hyaluronans.
 108. The kit according to claim 106, wherein the ECM component mixture comprises collagen and fibronectin. 